CN105154411A - Preparation methods of microbial dioxygenase crude enzyme and beta-ionone - Google Patents
Preparation methods of microbial dioxygenase crude enzyme and beta-ionone Download PDFInfo
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- CN105154411A CN105154411A CN201510462184.8A CN201510462184A CN105154411A CN 105154411 A CN105154411 A CN 105154411A CN 201510462184 A CN201510462184 A CN 201510462184A CN 105154411 A CN105154411 A CN 105154411A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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Abstract
The invention discloses a preparation method of a microgram-derived dioxygenase crude enzyme. The preparation method comprises the following steps: selecting scattered pantoea single colonies from a solid plate, inoculating the scattered pantoea single colonies to a seed solution culture medium, and carrying out shaking culture for 10-18 h under the condition that the pH is 6.4, the temperature is 28 DEG C, and the speed is 150r/min, and thus obtaining a seed solution; inoculating the seed solution to a fermentation culture medium according to the inoculation quantity of 2vol%, and thus obtaining a fermentation culture liquid; carrying out centrifugation on the fermentation culture for 30 min at the temperature of 4 DEG C and under the speed of 6000r/min, and collecting the supernate of the fermentation liquid, and thus obtaining the crude enzyme. The dioxygenase crude enzyme preparation is prepared through the microbial fermentation, and the dioxygenase crude enzyme is utilized for cracking lutein to produce flavor component beta-ionone. The whole technological process is simple, the cost is low, and the product beta-ionone obtained by catalytic synthesis is widely used in the industries of food and tobacco.
Description
Technical field
The present invention relates to a kind of preparation method of crude zyme preparation of microorganism dioxygenase, and utilize this crude enzyme liquid degraded xenthophylls to produce fragrance material β jononeionone.
Background technology
Dioxygenase be a class can make two Sauerstoffatoms in oxygen molecule all with the oxygenase of Binding Capacity, usual catalysis double bond and phenyl ring split relevant reaction.Existing dioxygenase can derive from plant, bacterium or fungi.But the dioxygenase of catalytic pyrolysis carotenoid is reported in plant more, but rarely has and be reported in microorganism.Mainly comprised five large classes by the dioxygenase reported in a large number in plant: CCD1, CCD4, CCD7 and CCD8 and NCEDs, the dioxygenase of these types can produce apocarotenoid and aldehyde material by catalytic pyrolysis carotenoid.Most product belongs to fragrance matter, as 3-hydroxyl β jononeionone, safranal, geranyl acetone and jononeionone etc.These fragrance matters have important using value in the field such as food, tobacco industry.Therefore develop this type of zymin and can provide wide application prospect for industrial production " natural " fragrance matter.
But the plant culturing cycle is long, dioxygenase expression amount is inadequate, cannot have in industrial production.Dioxygenase gene in plant is cloned in microorganism by part bibliographical information, and attempts vivoexpression.But eukaryotic gene expression amount in prokaryotic micro-organisms is inadequate, is difficult to realize scale operation.This research directly obtains by fermentable can the dioxygenase of cracking xenthophylls, can realize obtaining dioxygenase crude enzyme liquid in a large number, be applied to industrial production.Meanwhile, can generate β jononeionone after obtaining dioxygenase crude enzyme liquid cracking xenthophylls, be a kind of important fragrance matter.
β jononeionone is extract before a kind of important fragrance material and medicine, at room temperature has a kind of special aroma, can be widely used in food and tobacco industry.Simultaneously β jononeionone also shows the biological activitys such as antimicrobial, teratogenesis change, reducing blood-fat.β jononeionone can extract at occurring in nature and obtain from Activities of Some Plants, comprising violet, imperial Ting perfume and raspberry etc.The β jononeionone amount that these obtain from plants essential oil is micro-and very expensive, synthesizes at present mainly through chemical process.The synthesis step of chemical process is very complicated, and may produce environmental pollution and the wasting of resources, and the product of acquisition is difficult to be separated.Therefore the biosynthetic pathway of the alternative chemical synthesis process of exploitation is badly in need of.Consult current document, do not have pertinent literature report to adopt microbial enzyme to carry out biocatalysis synthesis β jononeionone.Method disclosed by the invention has important application prospect in β jononeionone industrial production.
Summary of the invention
Object of the present invention utilizes microorganism growth process to produce dioxygenase, and whole process is simple and quick, can obtain a large amount of dioxygenase crude enzyme liquids at short notice, facilitate industrial production to use.The crude enzyme liquid simultaneously obtained can produce β jononeionone by cracking xenthophylls, and the transformation efficiency of xenthophylls is 70%, is 0.02g/L by the concentration of β jononeionone in the final enzymatic reaction solution of vapor detection.
Technical scheme of the present invention is:
A preparation method for microorganism dioxygenase crude enzyme liquid, its step is as follows:
(1) disperse the single bacterium colony of general bacterium from picking solid plate, be inoculated on seed liquor substratum, at pH=6.4,28 DEG C, shaking culture 10-18h under the condition of 150r/min, obtains seed liquor;
(2) by seed liquor by volume 2% inoculum size be inoculated into from seed liquor in fermention medium, be placed in 28 DEG C of shaking tables, under the condition of pH=6.4,150r/min, cultivate 96-100h, obtain fermentation culture;
(3) by fermentation culture centrifugal 30min under 4 ° of C, 6000r/min, collect the supernatant liquor of fermented liquid, obtain crude enzyme liquid.
The formula of seed liquor substratum in described step (1): peptone 12.0g, yeast extract paste 6.0g, NaCl0.8g, distilled water 1L.
Fermented liquid culture medium prescription in described step (2): glucose 12.0g, yeast extract paste 6.0g, MgSO
40.15g, xenthophylls 0.02g, Tween-801.5ml, distilled water 1L.
The general bacterium of described dispersion is
pantoeadispersaaTCC29922.
Utilize microorganism dioxygenase crude enzyme liquid to prepare the method for alpha, beta-lonone, its step is as follows:
(1) xenthophylls is dissolved in the acetone of 20ml, makes the xenthophylls solution that concentration is 0.02g/L-1g/L, regulate pH to 6.4;
(2) get 1ml xenthophylls solution in test tube, add the 0.02mol/LNa of 3.5mLpH6.5
2hPO
4with 0.02mol/L citrate buffer solution, add the 0.05mol/LFeSO of 500ul
4solution, mixing, adds the crude enzyme liquid of 1ml, is placed in 35 DEG C of incubators, and lucifuge reaction 10min, obtains alpha, beta-lonone.
Beneficial effect of the present invention is: the present invention adopts microbial fermentation processes to obtain dioxygenase crude zyme preparation, and utilizes this crude enzyme liquid cracking xenthophylls to produce fragrance matter β jononeionone.Whole technical process is not only simple, and cost is low, and the product β jononeionone catalyzed and synthesized widely uses at food and tobacco industry.By the volume expansion of enzymatic reaction to 5L, in the solution after enzymatic reaction, alpha, beta-lonone concentration can reach 0.02g/L.
Accompanying drawing explanation
Fig. 1 is lutein degradation product GC-MS analysis chart.
Embodiment
Embodiment 1
From solid plate, picking disperses the single bacterium colony of general bacterium, is inoculated on seed liquor substratum, the formula of seed liquor substratum: peptone 12.0g, yeast extract paste 6.0g, NaCl0.8g, distilled water 1L, at pH=6.4,28 DEG C, shaking culture 10-18h under the condition of 150r/min, obtains seed liquor; By seed liquor by volume 2% inoculum size be inoculated into from seed liquor in fermention medium, fermented liquid culture medium prescription: glucose 12.0g, yeast extract paste 6.0g, MgSO
40.15g, xenthophylls 0.02g, Tween-801.5ml, distilled water 1L, be placed in 28 DEG C of shaking tables, under the condition of pH=6.4,150r/min, cultivate 96-100h, obtains fermentation culture; By fermentation culture centrifugal 30min under 4 ° of C, 6000r/min, collect the supernatant liquor of fermented liquid, obtain crude enzyme liquid, be stored in 4 DEG C.
Disperse general bacterium (
pantoeadispersa) being preserved in American Type Culture collection warehousing (Americantypeculturecollection), preserving number is ATCC29922.
Embodiment 2
Crude enzyme liquid cracking xenthophylls is utilized to produce the step of alpha, beta-lonone:
(1) reaction substrate xenthophylls adopts ethanol to dissolve, the dissolution process of concrete substrate: the xenthophylls taking 0.02g, be dissolved at 37 DEG C in the acetone of 20ml, after adopting Nitrogen evaporator to dry up, be that the potassium phosphate buffer constant volume of 6.4 is to 20ml with pH, regulate pH to 6.4, obtain xenthophylls solution, as substrate.Concentration of substrate is 0.02g/L-1g/L;
(2) thick enzyme reaction solution: get the xenthophylls solution in 1mL step (1) in the tool plug test tube of 50ml, add the 0.02mol/LNa of 3.5mLpH6.5
2hPO
4with 0.02mol/L citrate buffer solution, add the 0.05mol/LFeSO4 solution of 500ul, mixing, adds the crude enzyme liquid of 1ml, is placed in 35 DEG C of incubators, lucifuge reaction 10min;
(3) blank group 1 is set: replace the crude enzyme liquid in step (2) with the crude enzyme liquid boiling inactivation, other solution and reaction conditions constant;
(4) blank group 2 is set: replace the crude enzyme liquid in step (2) with the phosphoric acid buffer of pH6.4, other solution and reaction conditions constant;
(5) after reaction terminates, 3 group reaction liquid are all settled to 10mL, under spectrophotometer 448nm wavelength, survey absorbancy, calculate the amount of the rear xenthophylls of reaction.
(6) after the standardized solution of 6 gradients of the drafting preparation of xenthophylls typical curve carries out LC-MS analysis according to the chromatographic condition set up, with standard specimen peak area (A
i)/interior mark peak area (A
s) for ordinate zou (
y), mass concentration be X-coordinate (
x), drawing standard curve.
Lutein degradation rate calculation formula
(7), after determining the lutein content minimizing in thick enzyme reaction solution, adopted by reaction solution methylene dichloride to carry out extraction three times, carry out GC-MS assaying reaction product after utilizing Rotary Evaporators to carry out being concentrated to 1ml, see Fig. 1.
(8) gas phase (GC) condition is as follows: gas phase condition: chromatographic column: HP-5(30m × 0.25mm; 0.25rm); Heating schedule: initial temperature 45 DEG C, keeps 2min, is raised to 120 DEG C with 4 DEG C/min, retains 2min, is raised to 160 DEG C with 1 DEG C/min, retains 5min, is raised to 200 DEG C, is raised to 280 DEG C with 5 DEG C/min with 3 DEG C/min, retains 5min; Injector temperature: 270 DEG C; Transmission line temperature: 270 DEG C; Splitting ratio: 10:1; Carrier gas: He gas; Flow velocity: 1mL/min.
(9) mass spectrum (MS) condition: ionization mode EI, ionization voltage 70eV; Ion source temperature 230 DEG C; Mass scan range: 30-550amu.
Embodiment 3
Each material equal proportion contained by reaction solution in embodiment 2 being expanded 1000 times (end reaction volume is 5L) joins in the full-automatic medium-sized fermentor tank of 10L, in 28 DEG C, rotating speed is carry out lucifuge reaction 12h under 150r/min, utilizes 0.1mol/LHCl and 0.1mol/LNaOH solution full automatic control fermentor tank pH6.4.Measure absorb light by under 448nm after reaction terminates, the transformation efficiency calculating xenthophylls is 70%, is 0.02g/L by the concentration of alpha, beta-lonone in the final enzymatic reaction solution of vapor detection.
Claims (5)
1. a preparation method for microorganism dioxygenase crude enzyme liquid, is characterized in that, its step is as follows:
(1) disperse the single bacterium colony of general bacterium from picking solid plate, be inoculated on seed liquor substratum, at pH=6.4,28 DEG C, shaking culture 10-18h under the condition of 150r/min, obtains seed liquor;
(2) by seed liquor by volume 2% inoculum size be inoculated into from seed liquor in fermention medium, be placed in 28 DEG C of shaking tables, under the condition of pH=6.4,150r/min, cultivate 96-100h, obtain fermentation culture;
(3) by fermentation culture at 4 DEG C, centrifugal 30min under 6000r/min, collects the supernatant liquor of fermented liquid, obtains crude enzyme liquid.
2. the preparation method of microorganism dioxygenase crude enzyme liquid according to claim 1, is characterized in that: the formula of seed liquor substratum in described step (1): peptone 12.0g, yeast extract paste 6.0g, NaCl0.8g, distilled water 1L.
3. the preparation method of microorganism dioxygenase crude enzyme liquid according to claim 1, is characterized in that: the fermented liquid culture medium prescription in described step (2): glucose 12.0g, yeast extract paste 6.0g, MgSO
40.15g, xenthophylls 0.02g, Tween-801.5ml, distilled water 1L.
4. the preparation method of microorganism dioxygenase crude enzyme liquid according to claim 1, is characterized in that: the general bacterium of described dispersion is
pantoeadispersaaTCC29922.
5. utilize the microorganism dioxygenase crude enzyme liquid described in claim 1 to prepare the method for alpha, beta-lonone, it is characterized in that:
(1) xenthophylls is dissolved in the acetone of 20ml, makes the xenthophylls solution that concentration is 0.02g/L-1g/L, regulate pH to 6.4;
(2) get 1ml xenthophylls solution in test tube, add the 0.02mol/LNa of 3.5mLpH6.5
2hPO
4with 0.02mol/L citrate buffer solution, add the 0.05mol/LFeSO of 500ul
4solution, mixing, adds the crude enzyme liquid of 1ml, is placed in 35 DEG C of incubators, and lucifuge reaction 10min, obtains alpha, beta-lonone.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510462184.8A CN105154411A (en) | 2015-07-31 | 2015-07-31 | Preparation methods of microbial dioxygenase crude enzyme and beta-ionone |
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| CN201510462184.8A CN105154411A (en) | 2015-07-31 | 2015-07-31 | Preparation methods of microbial dioxygenase crude enzyme and beta-ionone |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282137A (en) * | 2016-08-02 | 2017-01-04 | 郑州轻工业学院 | The preparation method and applications of one Carotenoids 9,10 ' dioxygenase |
| CN115181737A (en) * | 2022-01-29 | 2022-10-14 | 广西中烟工业有限责任公司 | Lutein lyase, preparation method and method for synthesizing 3-hydroxy-beta-ionone |
| CN115426874A (en) * | 2020-04-21 | 2022-12-02 | 日本烟草产业株式会社 | Tobacco plant and method for producing same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396967A (en) * | 2013-08-09 | 2013-11-20 | 牛赡光 | Pantoea dispersa and application thereof |
-
2015
- 2015-07-31 CN CN201510462184.8A patent/CN105154411A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396967A (en) * | 2013-08-09 | 2013-11-20 | 牛赡光 | Pantoea dispersa and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| YUE ZHAO等: "Bioconversion of lutein to form aroma compounds by Pantoea dispersa", 《BIOTECHNOL LETT》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282137A (en) * | 2016-08-02 | 2017-01-04 | 郑州轻工业学院 | The preparation method and applications of one Carotenoids 9,10 ' dioxygenase |
| CN115426874A (en) * | 2020-04-21 | 2022-12-02 | 日本烟草产业株式会社 | Tobacco plant and method for producing same |
| CN115181737A (en) * | 2022-01-29 | 2022-10-14 | 广西中烟工业有限责任公司 | Lutein lyase, preparation method and method for synthesizing 3-hydroxy-beta-ionone |
| CN115181737B (en) * | 2022-01-29 | 2024-02-09 | 广西中烟工业有限责任公司 | Lutein lyase, preparation method and synthesis method of 3-hydroxy-beta-ionone |
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Application publication date: 20151216 |
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