CN105838626B - A kind of method of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde - Google Patents
A kind of method of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde Download PDFInfo
- Publication number
- CN105838626B CN105838626B CN201610296678.8A CN201610296678A CN105838626B CN 105838626 B CN105838626 B CN 105838626B CN 201610296678 A CN201610296678 A CN 201610296678A CN 105838626 B CN105838626 B CN 105838626B
- Authority
- CN
- China
- Prior art keywords
- eugenol
- gibberella fujikuroi
- conversion
- coniferyl aldehyde
- coniferyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of methods of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde, belong to field of biotechnology.The present invention is using eugenol as substrate, screening obtains the bacterial strain of one plant of the eugenol accumulation coniferyl aldehyde that can degrade, gibberella fujikuroi (Gibberella fujikuroi) ZH-34, on April 5th, 2016, it is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2016171, and preservation address is Wuhan, China Wuhan University.Coniferyl aldehyde is synthesized using bacterial strain conversion eugenol, when eugenol concentration is 1g/L, 0.96g/L coniferyl aldehyde, molar yield 94% can be produced；When gradation addition eugenol is converted, total eugenol concentration 6g/L can produce 3.76g/L coniferyl aldehyde, 1.35g/L coniferyl alcohol.The present invention provides the new methods that a kind of bioanalysis prepares coniferyl aldehyde.
Description
Technical field
The present invention relates to a kind of methods of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde, belong to biotechnology neck
Domain.
Background technique
Flavors and fragrances is widely used in food, feed, cosmetics, chemistry and field of medicaments.Many perfume (or spice) in the market
Material compound is still the extraction production through chemical method or from plant and animal source.Fragrance material master currently on the market
Will with chemical synthesis produce based on, but with people in substance production process concern and European countries and U.S. law
The fragrance material of requirement to food additives matter, biological fermentation process and enzymatic clarification, it is considered to be natural fragrance material, more
To be more concerned by people.Demand to market in 2011 to natural perfume material substance reaches 21,800,000,000 dollars.These natural perfume materials
Substance mainly passes through biosynthesis, plant method is extracted, enzyme and microorganism conversion obtain.Several different phenol type substances, such as fourth
Fragrant phenol, isoeugenol, ferulic acid all can serve as substrate, by the effect of microbial enzyme come synthetic perfume substance.Wherein, cloves
Phenol is the main component of caryophyllus oil, is obtained from extracting in plant clove of myrtaceae (Syzigium aromaticum), has and
The characteristics of source relatively wide cheap (existing market price is 5/Kg), market are easy to get, is recognized and is considered as one kind with using valence
The substrate of value.
Coniferyl aldehyde (coniferyl aldehyde), also known as ferulaldehyde, 4- hydroxy-3-methoxy cinnamic acid, molecular formula are
C10H10O3, be not readily dissolved in water, sterling be it is yellowish powdered, be present in confierophyte tree timber quality, it is wooden for biosynthesis
The intermediate of element, coniferyl aldehyde are used as deodorant tune, antifungal agent；Prostaglandin synthesis inhibitors；Chemical synthesis mainly has
It obtains by vanillic aldehyde (vanillin) and acetaldehyde condensation or with acid or caustic digestion lignin, is synthesized using microorganism conversion eugenol
The method of coniferyl aldehyde have not been reported.
Early in 1977, people began to the microorganism of research degradation eugenol.Japanese scholars Tadasa has found one plant of stick
The ability that there is bacillus (Corynebacterium sp.) degradation eugenol to generate coniferyl alcohol, ferulic acid and vanillic aldehyde；1996
Year, the method that French Rabenhorst uses batch culture using pseudomonad (Pseudomonas sp.) obtains 3.22g/
The coniferyl alcohol of L, the vanillic acid of 3.25g/L, the ferulic acid of 5.8g/L.1999, Japanese Furukawa etc. used fungi true yellow silk
Clothing mould (ByssochlamysfulvaV107) carries out conversion as substrate using eugenol and obtains the coniferyl alcohol of 21.7g/L, and has
The generation of a small amount of coniferyl aldehyde, ferulic acid and vanillic acid.2003, Japanese Furukawa etc. filtered out Pseudomonas fluorescens
(Pseudomonasjluorescens E118), the bacterium can generate 6.1g/L ferulic acid using eugenol；2010, India
Kadakol etc. is filtered out one plant of bacillus cereus (Bacillus cereus PN24), after addition eugenol conversion, transformant
Guaiacol, vanillic aldehyde, vanillic acid, protocatechuic acid intermediate metabolites can be detected in system；2011, Iranian Ashengroph etc.
Pseudomonas resinovorans (Pseudomonas resinovorans SPR1) bacterial strain is obtained through separation, eugenol can be converted and obtained
Obtain the vanillic aldehyde of 0.24g/L；2014, Lithuania Giedraityte etc. was separated to thermophilic molten Soil Bacillus
There is degradation eugenol to generate coniferyl alcohol, ferulic acid, vanillic aldehyde, perfume (or spice) for (Geobacillus sp.AY 946034) bacterial strain, the bacterium
The ability of oxalic acid.The metabolite of eugenol degradation involved in these researchs has coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillic aldehyde, perfume (or spice)
Oxalic acid, protocatechuic acid etc., and study majority and concentrate on analysis of the bacterium to eugenol katabolism approach, for the report of fungi
Road is relatively fewer.
Summary of the invention
Present invention firstly provides the microbial strains that a plant height produces coniferyl aldehyde.
The microbial strains are gibberella fujikuroi (Gibberellafujikuroi) ZH-34, on April 5th, 2016,
It is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2016171, and preservation address is Wuhan, China
Wuhan University.
The gibberella fujikuroi can convert eugenol and sequentially generate coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillic aldehyde, but accumulate
Coniferyl aldehyde is most.
The gibberella fujikuroi is 25~30 DEG C on Czapek's medium, after cultivating 5~7d, the white lint shape in bacterium colony surface,
Bottom shows slightly light cyan, and bacterium colony center is homogeneous fine and close velvet-like in satin, and bacterium colony edge is sparse, there is cyan slightly；Optics is aobvious
Micro- microscopic observation, thallus are mycelium, and vegetative hyphae has tabula, are able to carry out gemmation, and conidiophore is from mycelia top
Bear short and small, simply, top is tapered, spore Dan Sheng, and it is round, it has no and concatenates.
The present invention also provides the methods of application gibberella fujikuroi production coniferyl aldehyde, are with gibberella fujikuroi CCTCC NO:
The full cell of M2016171 or the enzyme of its production are catalyst, and using eugenol as substrate, building transformation system produces coniferyl aldehyde.
In one embodiment of the invention, gibberella fujikuroi thallus is added in cloves phenol solution, 30~35 DEG C, 80
24~60h of~180r/min concussion conversion.The thallus can be wet thallus or thallus pulvis.
In one embodiment of the invention, gibberella fujikuroi seed liquor is trained by 6~10% inoculum concentration access fermentation
Feeding base, 28~30 DEG C, 80~180r/min shake culture 18~for 24 hours, and addition substrate eugenol, 30~35 DEG C, 80~180r/
24~60h of min concussion conversion.
In one embodiment of the invention, gibberella fujikuroi seed liquor is trained by 6~10% inoculum concentration access fermentation
Feeding base, 28~30 DEG C, 80~180r/min shake culture 18~for 24 hours, disposably add the cloves of final concentration of 0.5~1.5g/L
Phenol, 30~35 DEG C, 80~180r/min concussion conversion, 24~60h.
In one embodiment of the invention, gibberella fujikuroi seed liquor is trained by 6~10% inoculum concentration access fermentation
Feeding base, 28~30 DEG C, 80~180r/min shake culture 18~for 24 hours, the eugenol of final concentration of 0.5~1.5g/L is first added,
30~35 DEG C, 80~180r/min, 6~8h is converted, adds eugenol at interval of 6~8h later；Eugenol adds 4~6 times altogether,
Total addition level is 2~9g/L.
In one embodiment of the invention, the eugenol is added to transformation system in the form of eugenol emulsion
In.
In one embodiment of the invention, the eugenol is added to transformation system in the form of eugenol emulsion
In, the preparation of eugenol emulsion: the Tween-80 of 0.5~1ml is added in 50~100ml water, and 115~121 DEG C of sterilizings 20~
After 30min, the eugenol of 1~2ml is added, 80~150r/min concussion 4~12h of emulsification is prepared into milky eugenol cream
Turbid.
In one embodiment of the invention, the fermentation medium contains: 5~20g/L of glucose, 2~1g/ of yeast extract
L, K2HPO41~5g/L, MgSO4 7H2O 0.2~1.0g/L, FeSO4·7H2O 0.1~0.2mg/L, GaCl2·2H2O 0.1
~0.4mg/L, H3BO30.1~0.3mg/L, CuSO4·5H2O 0.05~0.1mg/L of 0.02~0.04mg/L, KI,
MnSO4·7H2O0.1~0.4mg/L, NaMoO4·2H2O 0~0.2mg/L, pH 6.5~7.5, liquid amount are 50~100ml/
500ml shaking flask, 115~121 DEG C, sterilize 20~30min.
The present invention obtains the gibberella fujikuroi of one plant of degradation eugenol high yield coniferyl aldehyde by screening, and conversion eugenol is raw
The molar yield for producing coniferyl aldehyde reaches 90% or more.The present invention provides the new methods that a kind of bioanalysis prepares coniferyl aldehyde.
Biomaterial preservation
Gibberella fujikuroi (Gibberellafujikuroi) ZH-34 is preserved in Chinese Typical Representative training on April 5th, 2016
Object collection is supported, deposit number is CCTCC NO:M 2016171, and preservation address is Wuhan, China Wuhan University.
Detailed description of the invention
The high performance liquid chromatography of Fig. 1 microbe conversion liquid；Scheme A mixed sample, a: vanillic acid；B: coniferyl alcohol；C: vanillic aldehyde；d:
Ferulic acid；E: coniferyl aldehyde；F: eugenol, retention time a:11.37min；b:14.32min；c:15.27min；d:16.31min；
e:22.86min；f:26.41min；Figure B fermentation conversion fluid sample, b: coniferyl alcohol；C: vanillic aldehyde；E: coniferyl aldehyde；F: eugenol is protected
Stay time b:14.32min；c:14.38min；e:22.85min；f:26.39min.
Specific embodiment
Screen thin layer chromatography used in strain: solvent used is n-hexane, chloroform, anhydrous ether, acetic acid second
Ester and glacial acetic acid (monitoring ferulic acid, vanillic acid and the vanillic aldehyde in biotransformation simultaneously referring to the TLC such as Li Yonghong method),
The method quickly can detect the conversion fluid of large batch of bacterial strain to be sieved in a relatively short period of time.
The high performance liquid chromatography testing conditions for the product that analysis strain conversion eugenol generates are: Detection wavelength 280~
300nm, 25~30 DEG C of column temperature, 10~20 μ L of sample volume, two kinds of mobile phase As (acetonitrile), Mobile phase B (0.1% acetic acid) carries out ladder
Degree elution, 0~4min10~15%A, 85~90% B, 0.8~1ml/min of flow velocity；4~20min 25~30%A, 70~
75% B, 0.8~1ml/min of flow velocity；20~30min 70~80%A, 20~30%B, 0.8~1ml/min of flow velocity；30~
35min10~15%A, 85~90% B, 0.8~1ml/min of flow velocity.
The high performance liquid chromatography and Mass Spectrometry testing conditions for the product that analysis strain conversion eugenol generates are: detection
200~600nm of wavelength, 40~45 DEG C of column temperature, 1~2 μ L of sample volume, two kinds of flowing A (acetonitrile), Mobile phase B (0.1% formic acid) into
Row gradient elution, 0~17min 5~10%A, 90~95%B, 0.3~0.5ml/min of flow velocity；17~20min 60~70%
A, 30~40%B, 0.3~0.5ml/min of flow velocity；20~22min, 90~100%A, 0~10%B, 0.2~0.3ml/ of flow velocity
min；22~25min 5~10%A, 90~95%B, 0.3~0.5ml/min of flow velocity.
The preparation of eugenol emulsion: the Tween-80 of 0.5~1ml is added in 50~100ml water, and 115~121 DEG C go out
After 20~30min of bacterium, the eugenol of 1~2ml is added, concussion (80~150r/min) emulsifies 4~12h, is prepared into milky
Eugenol emulsion.
Inclined-plane culture: the glucose of 1~10g/L, 100~250g/L of potato juice, agar 20~25g/L, 115~121
DEG C, sterilize 20~30min, and bevel inoculation, cultivates 5~7d by 25~30 DEG C.
The culture of seed: the glucose of 1~10g/L, 100~250g/L of potato juice, yeast extract 1~5g/L, 115~
121 DEG C, sterilize 20~30min, 25~30 DEG C, 90~110r/min after inoculation, culture 18~for 24 hours.
Fermentation medium: 5~20g/L containing glucose, yeast extract 2~1g/L, K2HPO41~5g/L, MgSO4 7H2O
0.2~1.0g/L, FeSO4·7H2O 0.1~0.2mg/L, GaCl2·2H2O 0.1~0.4mg/L, H3BO30.1~0.3mg/
L, CuSO4·5H2O0.02~0.04mg/L, KI 0.05~0.1mg/L, MnSO47H2O 0.1~0.4mg/L, NaMoO4·
2H2O 0~0.2mg/L, pH 6.5~7.5, liquid amount be 50~100ml/500ml shaking flask, 115~121 DEG C, sterilizing 20~
30min is inoculated with by 6~10% inoculum concentration, and 25~30 DEG C, 80~180r/min, culture 18~for 24 hours, add 0.5~
The eugenol of 1.5g/L, converts 24~72h by 30~35 DEG C, 80~180r/min.
Selective agar medium: ammonium nitrate 1.0g/L, epsom salt 0.5g/L, ammonium sulfate 0.5g/L, potassium dihydrogen phosphate 0.5g/
L, dipotassium hydrogen phosphate 1.5g/L, sodium chloride 0.5g/L, yeast extract 2.0g/L, natural pH.
Enriched medium: 5g/L yeast extract, 10g/L peptone, 10g/L glucose, natural pH.
The screening and identification of the degradation eugenol bacterial strain of embodiment 1
5 Xiamen of Fujian Province, the micro- Huaian of peace, Henan Xinxiang, Heilungkiang Fuyuan and jiangsu wuxi places are acquired respectively differently
3~10cm pedotheque of point, the soil sample of acquisition claim 3~6g to be added to 30~60ml, the physiology of 115~121 DEG C of sterilizings respectively
In salt water, 25~30 DEG C, 90~150r/min, concussion mixes 15~30min.Taken under aseptic condition the upper soil sample clear liquid of 3~6ml by
8~12% inoculum concentration is inoculated into the Selective agar medium containing eugenol emulsion, and 25~30 DEG C, 90~150r/min, choosing
After selecting 2~5d of culture, it is inoculated into the enriched medium containing eugenol by 4~10% inoculum concentration and carries out enrichment culture, 25
~30 DEG C, 90~150r/min, 2~5d is cultivated, it is flat to be respectively coated the Bacterial Plate containing eugenol, Gause I and Cai Shi
On plate culture medium, 25~30 DEG C of 3~5d of culture, whether there is or not the generations of bacterium colony for observation.There is bacterium colony to generate, the well-grown list of picking
Bacterium colony lines in the slant medium containing eugenol respectively, cultivates 2~5d, is transferred to 24 holes equipped with seed culture medium
In culture plate (seed culture medium is directly connect after the mould inclined-plane culture that laboratory saves), 25~30 DEG C, 90~150r/min, training
18~36h is supported, eugenol emulsion conversion 24~48h of culture of final concentration of 0.5~1.5g/L is then added, conversion terminates
Afterwards, TLC method detects conversion results, obtains the bacterial strain of 146 plants of eugenols that can degrade altogether, these bacterial strain shaking flasks are carried out secondary screening
Afterwards, the product that the analysis of high performance liquid chromatography Preliminary detection generates, finally obtains one plant of ZH-34 bacterium, can convert eugenol and successively give birth to
At coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillic aldehyde, but coniferyl aldehyde can be accumulated.
Bacterial strain ZH-34 carries out Morphological Identification: 25~30 DEG C on Czapek's medium, after cultivating 5~7d, bacterium colony surface is in white
Color lint shape, bottom show slightly light cyan, and bacterium colony center is homogeneous fine and close velvet-like in satin, and bacterium colony edge is sparse, there is blueness slightly
Color, under the microscope, thallus is mycelium to optical microphotograph, and vegetative hyphae has tabula, is able to carry out gemmation, conidiophore is certainly
Mycelia top is born short and small, and simply, top is tapered, spore Dan Sheng, round, is had no and is concatenated.
Thallus is applied on solid plate culture medium, 30 DEG C of culture 5-7d, after, it is tried according to fungal gene group Rapid extraction
The extraction of agent box progress fungal gene group.Select eucaryote ITS universal amplification primer (upstream primer ITS1:5 '-
TCCGTAGGTGAACCTGCGG-3'；Downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '), the area PCR amplification ITS
Domain, after 1% agarose gel electrophoresis detection.Sequencing result carries out Blast sequence alignment in GeneBank.The bacterium is through morphology
It is Gibberella fujikuroi (Gibberellafujikuroi) with molecular biology identification.
Embodiment 2 generates coniferyl aldehyde using Gibberellafujikuroi ZH-34 conversion eugenol
Bacterial strain ZH-34 is transferred in slant medium, 25~30 DEG C of 5~7d of culture are transferred in seed culture medium, 25
~30 DEG C, 90~110r/min, culture 18~for 24 hours, fermentation medium is transferred to by 6%~8% inoculum concentration, 25~30 DEG C,
90~110r/min, after culture 18~for 24 hours, be separately added into final concentration of 0.5,1.0,1.5,2.0, the eugenol of 2.5g/L (with
The form of the eugenol emulsion of concentration 20g/L is added), 30~35 DEG C, 90~110r/min, convert 50h；It is carried out after processing
HPLC testing result such as the following table 1, wherein the molar yield under the conditions of 1.0g/L eugenol is 94%.
1 concentration of substrate of table produces the influence of coniferyl aldehyde to G.fujikuroi ZH-34 conversion eugenol
| Concentration of substrate g/L | 0.5 | 1.0 | 1.5 | 2.0 | 2.5L |
| Coniferyl aldehyde g/L | 0.15 | 0.96 | 1.08 | 0.46 | 0.42 |
Influence of 3 temperature of embodiment to coniferyl aldehyde is produced
Bacterial strain ZH-34 is transferred in slant medium, 25~30 DEG C of 5~7d of culture are transferred in seed culture medium, 25
~30 DEG C, 90~110r/min, culture 18~for 24 hours, fermentation medium is transferred to by 6%~8% inoculum concentration, 25~30 DEG C,
90~110r/min, culture 18~for 24 hours after, the eugenol of final concentration of 1.0g/L is added (with the eugenol milkiness of concentration 20g/L
The form of liquid is added), respectively under the conditions of temperature is 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 90~110r/min, conversion
50h；HPLC testing result such as the following table 2 is carried out after processing, wherein the molar yield of 1.0g/L is 92% under the conditions of 35 DEG C.
2 temperature of table produces the influence of coniferyl aldehyde to G.fujikuroi ZH-34 conversion eugenol
| Temperature DEG C | 25 | 30 | 35 | 40 | 45 |
| Coniferyl aldehyde g/L | 0.62 | 0.92 | 0.94 | 0.88 | 0.63 |
Embodiment 4 generates coniferyl aldehyde using Gibberellafujikuroi ZH-34 interval addition eugenol
Bacterial strain ZH-34 is transferred in slant medium, 25~30 DEG C of 5~7d of culture are transferred in seed culture medium, 25
~30 DEG C, 90~110r/min, culture 18~for 24 hours, fermentation medium is transferred to by 6%~8% inoculum concentration, 25~30 DEG C,
The eugenol of final concentration of 0.5~1.5g/L is added (with the cloves of concentration 20g/L after culture 18~for 24 hours in 90~110r/min
The form of phenol emulsion is added), 30~35 DEG C, 90~110r/min, 6~8h is converted, adds eugenol at interval of 6~8h later
Emulsion adds 4~6 times altogether for converting, and eugenol total addition level is 2~9g/L, and conversion terminates that 1.2~3.75g/ can be obtained
The coniferyl aldehyde of L, the molar yield of coniferyl aldehyde are 38.2%~55%.
Embodiment 5
Bacterial strain ZH-34 is transferred in slant medium, 25~30 DEG C of 5~7d of culture are transferred in seed culture medium, 25
~30 DEG C, 90~110r/min, culture 18~for 24 hours, 8000rpm low-temperature centrifugation collects thallus, with the phosphate-buffered of pH7.0
Liquid will collect obtained thallus and prepare bacteria suspension by mass fraction 50%.50ml bacteria suspension is taken, 300 μ l (6g/L) cloves are added
150 μ l Tween-80s are added in phenol emulsion simultaneously, after 110rpm, 30 DEG C of concussion shaking table culture 7h, add 200 μ l's (4g/L)
Eugenol emulsion continues after converting 48h.HPLC detection will be carried out after 10 times of conversion fluid dilutions, the results show that free cell turns
Change eugenol and generates the coniferyl aldehyde of 4.77g/L and the vanillic aldehyde of 21mg/L.It is surplus that there are also the eugenols of 2g/L in transformation system at this time
Remaining, the molar yield of coniferyl aldehyde is 56.3%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of gibberella fujikuroi (Gibberella fujikuroi) ZH-34 is preserved in Chinese Typical Representative on April 5th, 2016
Culture collection, deposit number are CCTCC NO:M 2016171, and preservation address is Wuhan, China Wuhan University.
2. a kind of method using the production coniferyl aldehyde of gibberella fujikuroi described in claim 1, which is characterized in that be with gibberella fujikuroi
The full cell of CCTCC NO:M 2016171 or the enzyme of its production are catalyst, using eugenol as substrate, building transformation system production
Coniferyl aldehyde.
3. according to the method described in claim 2, it is characterized in that, gibberella fujikuroi thallus is added in cloves phenol solution, 30
~35 DEG C, 80~180r/min concussion conversion, 24~60h.
4. according to the method described in claim 2, it is characterized in that, gibberella fujikuroi seed liquor is connect by 6~10% inoculum concentration
Enter fermentation medium, 28~30 DEG C, 80~180r/min shake culture 18~for 24 hours adds substrate eugenol, and 30~35 DEG C, 80
24~60h of~180r/min concussion conversion.
5. according to the method described in claim 2, it is characterized in that, gibberella fujikuroi seed liquor is connect by 6~10% inoculum concentration
Enter fermentation medium, 28~30 DEG C, 80~180r/min shake culture 18~for 24 hours, disposably add final concentration of 0.5~
The eugenol of 1.5g/L, 30~35 DEG C, 80~180r/min concussion conversion, 24~60h.
6. according to the method described in claim 2, it is characterized in that, gibberella fujikuroi seed liquor is connect by 6~10% inoculum concentration
Enter fermentation medium, 28~30 DEG C, 80~180r/min shake culture 18~for 24 hours, first add final concentration of 0.5~1.5g/L's
Eugenol 30~35 DEG C, 80~180r/min, converts 6~8h, adds eugenol at interval of 6~8h later；Eugenol adds altogether
4~6 times, total addition level is 2~9g/L.
7. according to any method of claim 4-6, which is characterized in that the eugenol is in the form of eugenol emulsion
It is added in transformation system.
8. the method according to the description of claim 7 is characterized in that the preparation of the eugenol emulsion: 0.5~1ml's spits
Temperature -80 is added in 50~100ml water, after 115~121 DEG C of 20~30min of sterilizing, the eugenol of 1~2ml of addition, 80~
150r/min concussion 4~12h of emulsification, is prepared into milky eugenol emulsion.
9. according to any method of claim 4-6, which is characterized in that the fermentation medium contains: 5~20g/ of glucose
L, yeast extract 2~1g/L, K2HPO41~5g/L, MgSO4 7H2O 0.2~1.0g/L, FeSO4·7H20.1~0.2mg/L of O,
GaCl2·2H2O 0.1~0.4mg/L, H3BO30.1~0.3mg/L, CuSO4·5H2O 0.02~0.04mg/L, KI 0.05
~0.1mg/L, MnSO4·7H2O 0.1~0.4mg/L, NaMoO4·2H2O 0~0.2mg/L, pH 6.5~7.5, liquid amount
For 50~100ml/500ml shaking flask, 115~121 DEG C, sterilize 20~30min.
10. the application of gibberella fujikuroi described in claim 1, which is characterized in that the field of the application is food, feed, makeup
Product, chemistry and field of medicaments, for producing coniferyl aldehyde, coniferyl alcohol, ferulic acid, vanillic aldehyde.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610296678.8A CN105838626B (en) | 2016-05-06 | 2016-05-06 | A kind of method of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610296678.8A CN105838626B (en) | 2016-05-06 | 2016-05-06 | A kind of method of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105838626A CN105838626A (en) | 2016-08-10 |
| CN105838626B true CN105838626B (en) | 2019-06-07 |
Family
ID=56591492
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610296678.8A Active CN105838626B (en) | 2016-05-06 | 2016-05-06 | A kind of method of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105838626B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019995B (en) | 2019-12-31 | 2021-04-27 | 厦门欧米克生物科技有限公司 | Method for producing vanillin by fermentation with eugenol as substrate |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0576860A2 (en) * | 1992-06-17 | 1994-01-05 | Haarmann & Reimer Gmbh | Process for the production of coniferylaldehyde |
| CN103820375A (en) * | 2013-12-13 | 2014-05-28 | 安徽师范大学 | Engineering strain for biologically producing ferulic acid and establishing method of engineering strain |
| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN105441340A (en) * | 2016-01-07 | 2016-03-30 | 南京工业大学 | High-producing strain of GA (gibberellin) 4+7 and application of high-producing strain |
-
2016
- 2016-05-06 CN CN201610296678.8A patent/CN105838626B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0576860A2 (en) * | 1992-06-17 | 1994-01-05 | Haarmann & Reimer Gmbh | Process for the production of coniferylaldehyde |
| CN103820375A (en) * | 2013-12-13 | 2014-05-28 | 安徽师范大学 | Engineering strain for biologically producing ferulic acid and establishing method of engineering strain |
| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN105441340A (en) * | 2016-01-07 | 2016-03-30 | 南京工业大学 | High-producing strain of GA (gibberellin) 4+7 and application of high-producing strain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105838626A (en) | 2016-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gallois et al. | Influence of culture conditions on production of flavour compounds by 29 ligninolytic Basidiomycetes | |
| Samson et al. | Polyphasic taxonomy of the heat resistant ascomycete genus Byssochlamys and its Paecilomyces anamorphs | |
| Pane et al. | Disease suppressiveness of agricultural greenwaste composts as related to chemical and bio-based properties shaped by different on-farm composting methods | |
| CN105567580B (en) | One plant of production sophorolipid saccharomycete and the preparation method and application thereof | |
| CN102321563B (en) | Amycolatopsis sp. and method for preparing vanillin through whole-cell transformation of Amycolatopsis sp. | |
| CN103468585B (en) | Monascus strain and application thereof in preparing functional monascus | |
| CN103865855A (en) | Bacillus subtilis strain and applications thereof | |
| CN108949597B (en) | Saccharomyces cerevisiae KMLY1-2 and separation method and application thereof | |
| CN109370958A (en) | One plant increases the lactobacillus plantarum of 3- hydroxy-2-butanone content and its application in soy sauce | |
| CN106987531B (en) | Wine membrana tectoria yeast and application thereof in monoterpene production | |
| Fagan et al. | Production of Hnalool, cis-and trans-nerolidol, and trans, trans-famesol by Saccharomyces fermentati growing as a film on· simulated wine | |
| CN108300677A (en) | One plant of streptomyces albus and its application in preparing microbialpreservatives | |
| De Aráujo et al. | Production of coconut aroma by fungi cultivation in solid-state fermentation | |
| CN105838626B (en) | A kind of method of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde | |
| Grondin et al. | Flavour production by Saprochaete and Geotrichum yeasts and their close relatives | |
| EP3339443B1 (en) | Streptomyces psammoticus omk-4 and method for producing vanillin | |
| Overy et al. | New Penicillium species associated with bulbs and root vegetables | |
| CN107893033A (en) | Aspergillus fumigatus SQH4 and the application in biotransformation method prepares texifolin | |
| CN104894173B (en) | A kind of preparation method of curcumin derivate | |
| Doster et al. | Description of a distinctive aflatoxin-producing strain of Aspergillus nomius that produces submerged sclerotia | |
| CN105779363B (en) | A kind of method of liquefied Serratia and its Synthesis heliotropin | |
| CN106479900B (en) | High yield monascus purpureus penicillium oxalicum Po-25 bacterial strain and application thereof | |
| Stchigel et al. | New species of Thielavia, with a molecular study of representative species of the genus | |
| CN112300944B (en) | Hordeum radiobacter strain and application thereof in preparation of fragrant substances | |
| CN104593267B (en) | Monascus purpureus and its application in 1 DNJ is prepared |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |