CN107236753A - The method that the construction method and conversion isoeugenol of isoeugenol monooxygenase activity aggregation produce vanillic aldehyde - Google Patents
The method that the construction method and conversion isoeugenol of isoeugenol monooxygenase activity aggregation produce vanillic aldehyde Download PDFInfo
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- 108030005392 Isoeugenol monooxygenases Proteins 0.000 title claims abstract description 89
- 238000004220 aggregation Methods 0.000 title claims abstract description 59
- 230000002776 aggregation Effects 0.000 title claims abstract description 59
- 230000000694 effects Effects 0.000 title claims abstract description 49
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 28
- BJIOGJUNALELMI-UHFFFAOYSA-N trans-isoeugenol Natural products COC1=CC(C=CC)=CC=C1O BJIOGJUNALELMI-UHFFFAOYSA-N 0.000 title claims abstract description 27
- BJIOGJUNALELMI-ONEGZZNKSA-N Isoeugenol Natural products COC1=CC(\C=C\C)=CC=C1O BJIOGJUNALELMI-ONEGZZNKSA-N 0.000 title claims abstract description 26
- BJIOGJUNALELMI-ARJAWSKDSA-N cis-isoeugenol Chemical compound COC1=CC(\C=C/C)=CC=C1O BJIOGJUNALELMI-ARJAWSKDSA-N 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 20
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01319—Isoeugenol synthase (1.1.1.319)
Abstract
The invention discloses the method that a kind of construction method of isoeugenol monooxygenase activity aggregation and conversion isoeugenol produce vanillic aldehyde, construction method includes S1, builds recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation：Small peptide 18A with amphipathic self-assembling function is connected with target gene isoeugenol monooxygenase, the active aggregations of isoeugenol monooxygenase 18A are built, and in expression in escherichia coli, obtain recombination bacillus coli；S2, the recombination bacillus coli production isoeugenol monooxygenase activity aggregation using structure：The recombination bacillus coli of structure is subjected to culture optimization, induction, the thalline after induction is collected by centrifugation, smudge cells is to obtain crude enzyme liquid, and gained crude enzyme liquid is isoeugenol monooxygenase activity aggregation.The present invention realizes the efficient production of vanillic aldehyde, is easy to industrial production application.
Description
Technical field
The present invention relates to the structure of the genetic engineering bacterium of isoeugenol monooxygenase and its biocatalysis technology field, especially
It is related to a kind of method that the construction method and conversion isoeugenol of isoeugenol monooxygenase activity aggregation produce vanillic aldehyde.
Background technology
Vanillic aldehyde is one of spices for having the call in the world, is widely used in food, beverage, medicine, cosmetics etc.
Industry.The vanillic aldehyde of current commercial type is divided into two kinds of natural perfume oxalaldehyde and chemical synthesis vanillic aldehyde.Wherein, chemical synthesis is fragrant
Oxalaldehyde accounts for more than the 90% of the market share.Study and find, the edible chemical synthesis vanillic aldehyde of heavy dose can cause dizziness, vomit, very
To damage internal organ.Natural vanillic aldehyde extracts from the beanpod of plant vanilla, because vanilla plantation by weather, the place of production,
Fang Zhengce etc. influence, annual output is less than 20t, and far from the demand for meeting people, and price is costly, per kilogram price
It is tens times of chemical synthesis vanillic aldehyde.The vanillic aldehyde obtained by biotransformation method belongs to natural perfume oxalaldehyde, its fragrance and day
Right vanillic aldehyde is as good as, and is one of most promising alternative with the cycle is short, the low advantage of production cost.Isobutyl
It is fragrant phenol wide material sources, cheap, it is the desired precursor material that microbe transformation method obtains vanillic aldehyde.Isoeugenol monooxygenase
It is the unique key enzyme that efficient catalytic isoeugenol obtains vanillic aldehyde.
Living things catalysis production vanillic aldehyde needs one of crucial problem solved is how efficiently to prepare isoeugenol list oxygenation
Enzyme.The core of enzyme technology is the efficient production, separation, formulation and application and development of enzyme, but the production of enzyme preparation at present
Journey is complicated, cost is very high, it has also become the restriction wide variety of key issue of enzymatic.In recent years, it has been found that by will be some
With amphipathic self-assembling function small peptide (Self-assembly peptides, SAPs, it is typically residual in 8-18 amino acid
Base) be connected to lyoenzyme end can effectively induced activity enzyme aggregate generation.Combined using active enzyme aggregate
Investment or cross-linking enzyme aggregation method (cross-linked enzyme aggregates, CLEA), which prepare immobilised enzymes, has operation
Simply, active high advantage, can significantly reduce protein immobilization cost, simplify technique, subsequently can also be successfully applied to
The preliminary purification of destination protein, is to realize that organized enzyme prepares industry and amplifies the basis purified with high flux at present, not yet there is structure
Go out any disclosure and report of isoeugenol monooxygenase activity aggregation.
The content of the invention
The technical problem to be solved in the present invention is that there is provided a kind of structure side of isoeugenol monooxygenase activity aggregation
The method that method and conversion isoeugenol produce vanillic aldehyde.
The technical solution adopted for the present invention to solve the technical problems is：A kind of isoeugenol monooxygenase that builds is provided to live
The method of property aggregation and its immobilization, comprises the following steps：
S1, build recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation：
By the small peptide 18A (EWLKAFYEKVLEKLKELF) with amphipathic self-assembling function and target gene isoeugenol
Monooxygenase is connected, and builds the active aggregations of isoeugenol monooxygenase -18A, and in expression in escherichia coli, obtain restructuring big
Enterobacteria；
S2, the recombination bacillus coli production isoeugenol monooxygenase activity aggregation using structure：
The recombination bacillus coli of structure is subjected to culture optimization, induction, the thalline after induction is collected by centrifugation, is crushed thin
Gained crude enzyme liquid is centrifuged and/or filtered to obtain crude enzyme liquid by born of the same parents, and it is the aggregation of isoeugenol monooxygenase activity to obtain precipitation
Body.
Preferably, step S1 comprises the following steps：
S1-1, bacterial strain E.coli BL21 (DE3) IEM and E.coli BL21 (DE3) pET30a-LipA-18A trained
Support, E. coli BL21 (DE3) IEM and E. coli BL21 (DE3) pET30a-LipA- is obtained respectively
18A；
S1-2, the gene order according to isoeugenol monooxygenase, design four pairs of PCR primers, including sense primer A, under
Swim primer B1, anti-sense primer B2, anti-sense primer B3 and anti-sense primer B4；Wherein sense primer A and anti-sense primer B4 is to include enzyme
The specific primer of enzyme site；
S1-3, in sequence respectively using four pairs of PCR primers, using E. coli BL21 (DE3) IEM as first
Secondary pcr template enters performing PCR amplification, and obtained PCR primer takes 1 μ L as the template of PCR next time after diluting 1000 times；Next time
PCR expanded using last PCR primer as template, finally give PCR primer target gene isoeugenol monooxygenase,
It is named as IEM-1；
S1-4, the target gene IEM-1 is purified；
S1-5, the extraction plasmid pET30a-LipA- from E. coli BL21 (DE3) pET30a-LipA-18A
18A；
S1-6, double digestion is carried out to the target gene IEM-1 and plasmid pET30a-LipA-18A respectively；
S1-7, the double digestion product progress nucleic acid electrophoresis by target gene IEM-1 and plasmid pET30a-LipA-18A, are returned
Receive double digestion product；
S1-8, the product after target gene IEM-1 and plasmid pET30a-LipA-18A double digestion recyclings connected
Connect, obtain the active aggregations of connection product isoeugenol monooxygenase -18A, be named as pET30a-IEM-18A；
S1-9, with the connection product pET30a-IEM-18A convert E. coli BL21 (DE3), obtain weight
Group bacterium E.coli BL21 (DE3) pET30a-IEM-18A.
Preferably, in step S1-2：
Sense primer A sequence is as follows：5’-tagctacatatgatggcaacgtttaccgcaatgatc-3’
Anti-sense primer B1 sequence is as follows：5’-gcggccgccttatctctcgaggttcttagactgccaac-3’
Anti-sense primer B2 sequence is as follows：5’-gtgcaacgctgctcggagacggaagggtagctt-3’
Anti-sense primer B3 sequence is as follows：5’-gactgccaacaaccgtgcaacgctgctcggaga-3’
Anti-sense primer B4 sequence is as follows：5’-tagctaaagctttcagttcttagactgccaacaaccgtgc-3’.
Preferably, step S2 comprises the following steps：
S2-1, the recombination bacillus coli of structure is inoculated in received containing card mycin liquid LB medium in cultivate to obtain
Obtain seed liquor；
S2-2, take the seed liquor of acquisition be inoculated in received containing card mycin solid-state LB culture mediums in cultivate；
S2-3, in the solid-state LB culture mediums add derivant (IPTG) induced；
The thalline after induction is collected by centrifugation in S2-4, low-temperature and high-speed, and thalline is resuspended with Glycine-NaOH cushioning liquid,
It is made into cell suspension, smudge cells；
S2-5, centrifugation, add isometric Glycine-NaOH or sodium carbonate-sodium hydroxide buffer in sediment
Solution, soft to be resuspended and (inhaled and beaten 2-3 times with liquid-transfering gun), the crude enzyme liquid of acquisition, centrifugation and/or filtering, it is isobutyl to obtain precipitation
Fragrant monooxyganase activity aggregation.
Preferably, the construction method is further comprising the steps of：
S3, isoeugenol monooxygenase activity aggregation immobilization：
Pass through cross-linked enzyme aggregate technology, using glutaraldehyde as cross linker, the isoeugenol list oxygenation that step S2 is obtained
Enzymatic activity aggregation carries out carrier-free immobilization.
Preferably, step S3 comprises the following steps：
Glutaraldehyde solution (concentration 100mmol/L is added dropwise in S3-1, the crude enzyme liquid obtained at low temperature toward step S2；Crosslinking
Time is 60-240 minutes), stirring；
S3-2, at low temperature high speed centrifugation, the sediment of acquisition are assembled for the isoeugenol monooxygenase activity of immobilization
Body, i.e. immobilised enzymes；
S3-3, by sodium carbonate-sodium hydroxide buffer solution immobilised enzymes is resuspended, cleaning immobilised enzymes surface attachment
Glutaraldehyde；
S3-4, low-temperature and high-speed centrifugation, immobilised enzymes is resuspended using sodium carbonate-sodium hydroxide buffer solution.
The present invention also provides the side that a kind of isoeugenol monooxygenase activity aggregation conversion isoeugenol produces vanillic aldehyde
Method, comprises the following steps：
T1, build recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation：
By the small peptide 18A (EWLKAFYEKVLEKLKELF) with amphipathic self-assembling function and target gene isoeugenol
Monooxygenase is connected, and builds the active aggregations of isoeugenol monooxygenase -18A, and in expression in escherichia coli, obtain restructuring big
Enterobacteria；
T2, the recombination bacillus coli conversion isoeugenol production vanillic aldehyde using structure：
The recombination bacillus coli of structure is subjected to culture optimization, induction, the thalline after induction is collected by centrifugation, bacterium is resuspended
Body is made into cell suspension, adds isoeugenol and is converted, obtains the reaction solution containing vanillic aldehyde.
Preferably, step S1 comprises the following steps：
T1-1, bacterial strain E.coli BL21 (DE3) IEM and E.coli BL21 (DE3) pET30a-LipA-18A trained
Support, E. coli BL21 (DE3) IEM and E. coli BL21 (DE3) pET30a-LipA- is obtained respectively
18A cells；
T1-2, the gene order according to isoeugenol monooxygenase, design four pairs of PCR primers, including sense primer A, under
Swim primer B1, anti-sense primer B2, anti-sense primer B3 and anti-sense primer B4；Wherein sense primer A and anti-sense primer B4 is to include enzyme
The specific primer of enzyme site；
T1-3, in sequence respectively using four pairs of PCR primers, using E. coli BL21 (DE3) IEM as first
Secondary pcr template enters performing PCR amplification, and obtained PCR primer takes 1 μ L as the template of PCR next time after diluting 1000 times；Next time
PCR expanded using last PCR primer as template, finally give PCR primer target gene isoeugenol monooxygenase,
It is named as IEM-1；
T1-4, the target gene IEM-1 is purified；
T1-5, the extraction plasmid pET30a-LipA- from E. coli BL21 (DE3) pET30a-LipA-18A
18A；
T1-6, double digestion is carried out to the target gene IEM-1 and plasmid pET30a-LipA-18A respectively；
T1-7, the double digestion product progress nucleic acid electrophoresis by target gene IEM-1 and plasmid pET30a-LipA-18A, are returned
Receive double digestion product；
T1-8, the product after target gene IEM-1 and plasmid pET30a-LipA-18A double digestion recyclings connected
Connect, obtain the active aggregations of connection product isoeugenol monooxygenase -18A, be named as pET30a-IEM-18A；
T1-9, with the connection product pET30a-IEM-18A E. coli BL21 (DE3) is transferred to, obtains weight
Group bacterium E.coli BL21 (DE3) pET30a-IEM-18A.
Preferably, in step T1-2：
Sense primer A sequence is as follows：5’-tagctacatatgatggcaacgtttaccgcaatgatc-3’
Anti-sense primer B1 sequence is as follows：5’-gcggccgccttatctctcgaggttcttagactgccaac-3’
Anti-sense primer B2 sequence is as follows：5’-gtgcaacgctgctcggagacggaagggtagctt-3’
Anti-sense primer B3 sequence is as follows：5’-gactgccaacaaccgtgcaacgctgctcggaga-3’
Anti-sense primer B4 sequence is as follows：5’-tagctaaagctttcagttcttagactgccaacaaccgtgc-3’.
Preferably, step T2 comprises the following steps：
T2-1, the recombination bacillus coli of structure is inoculated in received containing card mycin seed culture medium in cultivate to obtain
Seed liquor；
T2-2, obtain seed liquor be inoculated in received containing card mycin fermentation medium in culture；
T2-3, in the fermentation medium add derivant (IPTG) induced；
T2-4, by the zymotic fluid low-temperature centrifugation after induction, collect thalline, be resuspended with Glycine-NaOH cushioning liquid
Thalline, is made into cell suspension；
T2-5, take cell suspension, add isoeugenol and converted, reaction solution of the acquisition containing vanillic aldehyde.
Beneficial effects of the present invention：By isoeugenol monooxygenase gene and the amphipathic small peptide with self-assembling function
18A constructs isoeugenol monooxygenase activity aggregation, centrifugation, it is broken obtain aggregation, without cumbersome purification step.It is logical
The isoeugenol monooxygenase for crossing carrier-free immobilization can be directly used for conversion isoeugenol production vanillic aldehyde, reduce enzyme purification step
Suddenly, production cost is saved, the efficient production of vanillic aldehyde is realized, is easy to industrial production application.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used, is to be unless otherwise specified in following embodiments
It is commercially available from routine biochemistry reagent shop.Quantitative test in following examples, is respectively provided with three repetition experiments, as a result takes
Average value.
The construction method of the isoeugenol monooxygenase activity aggregation of the present invention, step includes as follows：
First, the structure of recombinant bacterium：Build the recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation
Wherein, bacterial strain：By E.coli BL21 (DE3) IEM of Shenzhen University's preservation, (deposit number is CGMCC
) and E.coli BL21 (DE3) pET30a-IEM-18A No.8918；It is biological that E. coli BL21 (DE3) is purchased from raw work
Engineering (Shanghai) Co., Ltd..E.coli BL21 (DE3) pET30a-IEM-18A belongs to ETEC (Escherichia
Coli), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on December 26th, 2016 (referred to as
CGMCC, address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.13493.
The preparation of conventional solution and culture medium：
(1) ampicillin (100mg/mL):5g ampicillins are weighed, are dissolved with sterilized water, constant volume is in 50mL capacity
Bottle, is filtered with disposable filter, is sub-packed in 1.5mL EP pipes, -40 DEG C of preservations.
(2) card receives mycin (50mg/mL):Weigh 2.5g cards and receive mycin, dissolved with sterilized water, constant volume in 50mL volumetric flasks,
Filtered with disposable filter, be sub-packed in 1.5mL EP pipes, -40 DEG C of preservations.
(3) IPTG (isopropylthiogalactoside, 0.8mol/L)：0.2g IPTG are weighed, are dissolved with sterilized water, constant volume
In 10mL volumetric flasks, filtered with disposable filter, be sub-packed in 1.5mL EP pipes, -40 DEG C of preservations.
(4) nucleic acid electrophoresis cushioning liquid (1 × TAE)：50 × TAE storing liquids are diluted 50 times with ultra-pure water, protected under normal temperature
Deposit stand-by.
(5) 1% Ago-Gels are prepared：Weigh 0.25g agaroses and be dissolved in 1 × TAE of 25mL, microwave stove heat 1min is extremely
Glue is uniformly dissolved, and non-scald on hand is cooled at room temperature, adds 3 μ L nucleic acid dyes, is mixed, is poured into glue plate, cool down at room temperature standby
(agarose used is purchased from Beijing Quanshijin Biotechnology Co., Ltd).
(6)0.1mol/L CaCl2：Prepare 0.1mol/L CaCl2Solution, is filtered with disposable filter, is sub-packed in
1.5mL EP are managed, -20 DEG C of preservations.
(7) in protein electrophorese liquid preparation (1*SDS-PAGE electrophoretic buffers)：By 5*SDS-PAGE electrophoretic buffers
1*SDS-PAGE electrophoretic buffers are diluted to deionized water to can be used.
(8) 50% glycerine：Glycerine and sterilized water 1:1 mixing, 121 DEG C of autoclaving 20min, room temperature preservation is standby.
(9) liquid LB medium：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, use 5mol/L NaOH
The culture medium is adjusted to pH 7.0；121 DEG C of autoclaving 20min.
(10) solid-state LB culture mediums compound method：
1. prepare：121 DEG C of autoclaving 20min after 2g agar powders, heating for dissolving are added in 100mL LB culture mediums.
2. antibiotic is added：After solid medium autoclaving, culture medium is placed in aseptic operating platform, treats that culture medium is cold
Antibiotic is added when but to non-scald on hand, mixing is down flat plate, and each flat board pours into 15-20mL solid mediums.
3. preserve：With sealed membrane by plate seal, it is inverted, is stored in 4 DEG C of refrigerators.
4. antibiotic concentration：The final concentration of 100 μ g/mL of ampicillin in culture medium, card receives final concentration of 50 μ of mycin
g/mL。
1st, bacterial strain E.coli BL21 (DE3) IEM and E.coli BL21 (DE3) pET30a-LipA-18A culture：
(1) a small amount of E.coli BL21 (DE3) IEM is picked with pipette tips and is inoculated in 5mL liquid LB mediums (containing 100 μ g/mL
Ampicillin), in incubated overnight under 37 DEG C, 200rpm.
(2) a small amount of E.coli BL21 (DE3) pET30a-LipA-18A is picked with pipette tips and is inoculated in 5mL solid LB medias
(receiving mycin containing 50 μ g/mL cards), in incubated overnight under 37 DEG C, 200rpm.
2nd, according to design, it is necessary to introduce Nde I and Hind III at isoeugenol monooxygenase (IEM) gene two ends by PCR
Two restriction enzyme sites, due to, containing a restriction enzyme site of Hind III, therefore, not changing the amino acid of its coding in IEM genes
Under conditions of, Hind III some base, i.e. rite-directed mutagenesis in mutation IEM.According to IEM gene orders, devise four couples of PCR and draw
Thing, including sense primer A, anti-sense primer B1, anti-sense primer B2, anti-sense primer B3 and anti-sense primer B4；Sense primer A and downstream
Primer B4 is the specific primer comprising restriction enzyme site.
Wherein, sense primer A sequence is as follows：5’-tagctacatatgatggcaacgtttaccgcaatgatc-3’；
Anti-sense primer B1 sequence is as follows：5’-gcggccgccttatctctcgaggttcttagactgccaac-3’；
Anti-sense primer B2 sequence is as follows：5’-gtgcaacgctgctcggagacggaagggtagctt-3’；
Anti-sense primer B3 sequence is as follows：5’-gactgccaacaaccgtgcaacgctgctcggaga-3’；
Anti-sense primer B4 sequence is as follows：5’-tagctaaagctttcagttcttagactgccaacaaccgtgc-3’.
3rd, above-mentioned four pairs of primers are used respectively in sequence, to cultivate E. coli BL21 (DE3) IEM of acquisition
Enter performing PCR amplification for first time pcr template, obtained PCR primer takes 1 μ L as the template of PCR next time after diluting 1000 times,
PCR next time is expanded using last PCR primer as template later.Extension increasing sequence (the PCR primer purpose finally given
Gene isoeugenol monooxygenase) it is named as IEM-1.
Wherein, PCR reaction systems are：System total amount is 25 μ L, and wherein upstream and downstream primer (concentration is 100 μm of ol/L) is each
0.75 μ L, the μ L of template 1, the μ L of 2 × Pfu polymerase 12.5 are added, finally adds the μ L polishings of aqua sterilisa 10.Being loaded principle is：First add
The few reagent of amount, the big reagent of the amount of adding, it is therefore intended that the molecular agents rushed in even system；Template and polymerase finally add
Enter, whole process is maintained to be operated on ice, reduce too high infringement (the polymerase PrimeSTAR Max DNA used to enzyme of temperature
Polymerase is purchased from Takara companies).
PCR reaction conditions are：98 DEG C of pre-degenerations 2min, 98 DEG C of denaturation 10s, 60 DEG C of annealing 15s, 72 DEG C extend 10s, 72 DEG C
Continue to extend 10min, be finally reduced to 4 DEG C, you can take out.Four PCR are this reaction condition.
The PCR primer of last time is analyzed into it (nucleic acid used with agarose gel electrophoresis after four times PCR terminates
Molecular weight supermarker, Loading Buffer are Centrix Technology Ltd. purchased from Beijing health), and use gel imaging
System observes glue figure, and the rite-directed mutagenesis IEM molecular size ranges that as a result display is obtained by PCR are in 1.5kb or so, and the 4th step is obtained
PCR primer be bright single tape after agarose electrophoresis is verified.
4th, PCR primer target gene IEM-1 is carried out with DNA fragmentation purification kit (being purchased from Takara companies) pure
Change.
With mini-scale plasmid extracts kit (be purchased from Shanghai bioengineering Science and Technology Ltd.) from E. coli
Plasmid pET30a-LipA-18A is extracted in BL21 (DE3) pET30a-LipA-18A.
5th, product (target gene IEM-1) after purification and the vector plasmid pET30a-LipA-18A extracted distinguish
Double digestion is carried out with III two kinds of restriction enzymes of Nde I and Hind (being purchased from Beijing NEB Co., Ltds).
Double digestion total system is 50 μ L：Nde I 1 μ L, Hind III 1 μ L, DNA or the μ L of 1 μ L, 10x NEBuffer of plasmid 5, go out
Bacterium water polishing is to 50 μ L.Digestion condition：Double digestion is stayed overnight, 37 DEG C of digestion temperature.
6th, target gene IEM-1 and plasmid pET30a-LipA-18A double digestion product is subjected to nucleic acid electrophoresis, uses DNA
Glue reclaim kit (being purchased from Takara companies) reclaims double digestion product.
7th, the product after target gene IEM-1 and plasmid pET30a-LipA-18A double digestion recyclings is attached, will
The obtained active aggregations of connection product isoeugenol monooxygenase -18A, are named as pET30a-IEM-18A.
Connection total system is 20 μ L：Target gene IEM-11 μ L, plasmid pET30a-LipA-18A 8 μ L, 10x Buffer
2 μ L, T4DNA ligases (being purchased from Beijing NEB Co., Ltds) 1 μ L, the μ L of aqua sterilisa 8.Target gene IEM-1 and plasmid
PET30a-LipA-18A mol ratio is about 3:1, the gross mass of carrier and purpose fragment is in 0.01 μ g or so.Condition of contact is
16 DEG C, 4h, 4 DEG C are overnight.
8th, with connection product pET30a-IEM-18A Transformed E .coli BL21 (DE3) competent cell, recombinant bacterium is obtained
E.coli BL21(DE3)pET30a-IEM-18A。
9th, recombinant bacterium is verified by bacterium colony PCR and recombinant plasmid double digestion, and chooses positive clone molecule and carry out base
Because of sequencing.
E.coli BL21 (DE3) pET30a-IEM-18A sequencing results：
2nd, isoeugenol monooxygenase activity aggregation is produced using the recombinant bacterium of structure
The preparation of conventional solution and culture medium, with reference to above-mentioned recombinant bacterium structure in solution and culture medium preparation.
1st, take 50 μ L E.coli DL21 (DE3) pET30a-IEM-18A to be inoculated in 50mL liquid LB mediums and (contain 100 μ
G/mL cards receive mycin), in incubated overnight under 37 DEG C, 200rpm, produce seed liquor.
2nd, take the seed liquor of 1mL steps 1 to be inoculated in 100mL and receive the solid LB media of mycin containing 100 μ g/mL cards, in 37
DEG C, to cultivate to OD600 under 200rpm be 0.8, adds 0.8mmol/IPTG, 25 DEG C, 200rpm induces 16h.
3rd, 10000rpm low temperature (less than 4 DEG C) be collected by centrifugation induction after thalline, with the glycine of 0.05mol/L pH 10.5-
Thalline is resuspended in sodium hydroxide buffer solution, is made into 60g/L cell suspension (bacteria suspension), uses high pressure homogenizer smudge cells, if
Put pressure 800bar, 40mL bacteria suspension broken three minutes.
4th, 7000rpm centrifuges 5min, and supernatant is separately deposited, and isometric 0.05mol/L pH 10.5 is added in sediment sweet
Propylhomoserin-sodium hydroxide buffer solution, it is soft to be resuspended and (inhaled and beaten 2-3 times with liquid-transfering gun), gained crude enzyme liquid is centrifuged and/or filtered,
It is isoeugenol monooxygenase activity aggregation to obtain precipitation.
3rd, the immobilization of isoeugenol monooxygenase activity aggregation
Wherein, the preparation of culture medium：
Seed culture medium (g/L)：Tryptone 10, yeast extract 5, NaCl 10；PH 7.0, liquid amount 200mL/
500mL conical flasks.
Fermentation medium (g/L)：Tryptone 10, yeast extract 5, NaCl 10；PH 7.0, liquid amount 400mL/
1000mL conical flasks.
1st, cell culture
Seed culture：Under aseptic condition, 200 μ L E.coli are added in seed culture medium (receiving mycin containing 50 μ g/mL cards)
BL21 (DE3) pET30a-IEM-18A, in shaking incubated overnight under 37 DEG C, 200rpm, obtains seed liquor.
Fermented and cultured：Under aseptic condition, 80mL seed liquors are taken to be inoculated in fermentation medium (receiving mycin containing 50 μ g/mL cards)
In, when shaking culture under 37 DEG C, 200rpm to OD600 is 0.8, add 0.8mmol/L IPTG, 30 DEG C of induction 8h.
2nd, immobilised enzymes (the isoeugenol monooxygenase activity aggregation of immobilization) is prepared
After induction terminates, bacterium solution is centrifuged into 15min in 10000rpm under low temperature (less than 4 DEG C), wet thallus is collected, added
0.05mol/L pH 10.5 Glycine-NaOH cushioning liquid is made into 65g/L cell suspension, is crushed with homogenizer height
Broken cell suspension, it is 800bar, 40mL cell suspensions circulation 3min to set homogenizer pressure.
Bacterium solution is collected after broken end.At 4 DEG C, 10000rpm high speed centrifugation 15min, abandoning supernatant adds in precipitation
Enter 10.5 sodium carbonate of 10mL 0.05mol/L pH-sodium hydroxide buffer solution, soft that precipitation is resuspended, gained crude enzyme liquid is as different
Cloves monooxyganase activity aggregation.
Complete crude enzyme liquid will be resuspended the glutaraldehyde solutions of 121 μ L 50% are slowly added dropwise in (less than 4 DEG C) under low temperature, be added dropwise
Keep magnetic agitation uninterrupted simultaneously, low temperature is kept during stirring, causes enzyme to inactivate with high temperature-proof.Solution is taken out after stirring 2h；4
At DEG C, 10000rpm high speed centrifugation 15min, supernatant discarding takes 10.5 sodium carbonate of 20mL 0.05mol/L pH-sodium hydroxide to delay
Rush solution to be resuspended twice, glutaraldehyde of cleaning immobilised enzymes surface attachment etc.；(10000rpm) centrifugation at a high speed of (less than 4 DEG C) of low temperature
Supernatant discarding, prepares isoeugenol monooxygenase immobilised enzymes.
4th, using isoeugenol monooxygenase activity aggregation conversion isoeugenol production vanillic aldehyde
Embodiment 1
50 μ L are taken out from glycerol tube E.coli BL21 (DE3) pET30a-IEM-18A of -80 DEG C of preservations, according to above-mentioned kind
The cultural method overnight incubation of son culture, takes 1mL seed liquors to be inoculated in and receives the fermentation medium of mycin containing 50 μ g/mL cards, when
When OD600 is 0.8,0.8mmol/L IPTG are added, inducing temperature is 30 DEG C, and 200rpm induces 16h；By the fermentation after induction
(less than 4 DEG C) centrifugation 15min of liquid 10000rpm low temperature, supernatant discarding collects thalline, with 0.05mol/L pH 10.5 sweet ammonia
Thalline is resuspended in acid-sodium hydroxide buffer solution, is made into 80g/L cell suspension.
10mL thalline (cell suspension) are taken, 260mmol/L isoeugenols are added, in converting 48h under 25 DEG C, 200rpm, surveyed
The concentration for determining vanillic aldehyde in end reaction liquid is 2.24g/L.
Embodiment 2
50 μ L are taken out from glycerol tube E.coli BL21 (DE3) pET30a-IEM-18A of -80 DEG C of preservations, according to above-mentioned kind
The cultural method culture 14h of son culture, takes 1mL seed liquors to be inoculated in and receives the fermentation medium of mycin containing 50 μ g/mL cards, when
When OD600 is 0.8,0.8mmol/L IPTG are added, 30 DEG C induce, and 200rpm, induction time is 8h, by the zymotic fluid after induction
(less than 4 DEG C) centrifugation 15min of 10 000rpm low temperature, supernatant discarding collects thalline；With 0.05mol/L pH 10.5 sweet ammonia
Thalline is resuspended in acid-sodium hydroxide buffer solution, and high pressure smudge cells suspension collects precipitation, adds 10mL 0.05mol/L pH
10.5 sodium carbonate-sodium hydroxide buffer solution, it is soft to be resuspended, 121 μ L 50% penta 2 are slowly added dropwise in (less than 4 DEG C) under low temperature
Aldehyde solution, keeps centrifuging and taking after magnetic agitation 2h to precipitate, prepares isoeugenol monooxygenase immobilised enzymes.
Solution of the 9mL containing immobilised enzymes is taken, 1mL DMSO, 100mmol/L isoeugenol is added, 25 DEG C, 200rpm turns
Change 36h, reaction terminates rear centrifugal reaction solution, and supernatant discarding cleans cross-linking enzyme with cushioning liquid twice, then uses 9mL 0.05mol/
Cross-linking enzyme is resuspended in the Glycine-NaOH cushioning liquid of L pH 10.5, continues enzymic catalytic reaction next time, and Reusability is handed over
Join enzyme, determine the concentration of vanillic aldehyde in each reaction solution；As a result show that it possesses good operational stability, reuse 7 times
Enzyme activity is retained in more than 60% afterwards.
The assay method of vanillic aldehyde：
Vanillic aldehyde and isoeugenol are determined with high performance liquid chromatography (HPLC).
Sample treatment：Reaction solution adds the ethanol with reaction solution same volume, protein precipitation (isoeugenol monooxygenase activity
Aggregation), and substrate (isoeugenol) and product (vanillic aldehyde), 10000r/min centrifugation 5min are dissolved, then 10- is diluted with ethanol
20 times (10 times are diluted in embodiment), membrane filtration is to be measured.
Chromatographic column：250 × 4.6mm of Thermo BDS HYPERSIL C18 reverse-phase chromatographic columns；
Mobile phase：A=0.01% glacial acetic acid aqueous solutions, B=methanol；
Gradient elution program：35%B during beginning, is kept for 7 minutes, increases to 70%B during to 15min, be down to during to 17min
35%B；
Column temperature：30℃；
Ultraviolet detection at flow velocity 1mL/min, wavelength 280nm, the μ L of sample size 20.The wherein appearance of vanillic aldehyde and isoeugenol
Time is respectively 7.8min and 17.4min or so.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair
Equivalent structure or equivalent flow conversion that bright description is made, or directly or indirectly it is used in other related technology necks
Domain, is included within the scope of the present invention.
<110>Shenzhen University
<120>The method that the construction method and conversion isoeugenol of isoeugenol monooxygenase activity aggregation produce vanillic aldehyde
<130> CN1618492YZ
<160> 6
<210> 1
<211> 36
<212> DNA
<213>Artificial sequence
<400> 1
tagctacata tgatggcaac gtttaccgca atgatc 36
<210> 2
<211> 38
<212> DNA
<213>Artificial sequence
<400> 2
gcggccgcct tatctctcga ggttcttaga ctgccaac 38
<210> 3
<211> 33
<212> DNA
<213>Artificial sequence
<400>3
gtgcaacgct gctcggagac ggaagggtag ctt 33
<210> 4
<211> 33
<212> DNA
<213>Artificial sequence
<400>4
gactgccaac aaccgtgcaa cgctgctcgg aga 33
<210> 5
<211>40
<212> DNA
<213>Artificial sequence
<400>5
tagctaaagc tttcagttct tagactgcca acaaccgtgc 40
<210> 6
<211>40
<212> DNA
<213>Artificial sequence
<400>6
atggcaacgt ttgaccgcaa tgatccgcag ttggcaggaa cgatgttccc cacccgaata
gaggcgaatg tctttgacct tgaaattgag ggcgagatcc cacgtgcaat caacgggagc
ttcttccgca acacccccga acctcaggtc accacgcaac ctttccacac cttcatcgat
ggggatggtt tggcgtctgc ttttcatttc gaagatggcc aggtcgactt tgtcagccgt
tgggtatgta ctcctcgctt tgaagctgag cggtcggctc gtaaatcact cttcggtatg
taccgcaatc cgttcactga tgatccatcg gtagaaggta ttgatcgtac agtcgccaac
accagtatca tcactcatca cgggaaagta ctggccgcaa aggaagatgg actaccttat
gagcttgacc cccaaaccct ggaaacccga ggtcgttatg attacaaggg gcaggtaacc
agccatacac atacagcgca ccctaagttc gacccccaga caggtgaaat gttactcttc
ggctccgctg ctaaaggcga acgaacgctt gatatggcgt actatattgt tgatcgctac
ggcaaggtga cacatgagac ctggtttaag cagccttacg gtgcattcat gcacgacttt
gctgtcacgc gcaactggtc aatctttccg atcatgcctg cgacaaatag ccttgagcgt
cttaaagcaa agcagcccat ttacatgtgg gagcctgagc gaggaagcta tataggagta
cttcctcgtc gtggtcaggg caaggacatt cgttggttcc gtgccccggc gttgtgggtt
ttccatgtcg tgaatgcttg ggaggaaggg aatagaattc tgattgactt gatggaaagt
gagattttgc cgttcccatt cccgaactcg cagaaccttc catttgatcc ctccaaggct
gttccgcgtc taacccgttg ggaaattgat ctcaatagtg gtaacgatga gatgaaacgt
acgcagctac acgaatattt tgcagaaatg cctatcatgg atttccgttt tgcgctccag
gatcatcgct acgcctacat gggggttgac gatcctcgtc gccccttagc tcatcagcaa
gctgaaaaaa tctttgccta caattcgtta ggggtttggg acaaccatcg taaagattat
gaactttggt ttacgggaaa aatgtctgca gcgcaggaac cggcgtttgt tcctagaagc
ccagatgcgc ctgagggcga tggctaccta ctcagtgtag tagggcggct cgatgaagat
cgtagcgatc tagttatcct tgatacgcaa tgtttggcag ctgggcctgt ggccactgtc
aagctaccct tccgtctccg agcagcgttg cacggttgtt ggcagtctaa gaactgaaag
cttccgaccc caccgaccac gccaacgcca ccaaccaccc caaccccgac gccgctggaa
cttgaactga agttaaaact ggaattagaa ttaaagctga aa 1542
Claims (10)
1. a kind of construction method of isoeugenol monooxygenase activity aggregation, it is characterised in that comprise the following steps：
S1, build recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation：
Small peptide 18A with amphipathic self-assembling function is connected with target gene isoeugenol monooxygenase, isobutyl is built fragrant
Phenol monooxygenase -18A activity aggregations, and in expression in escherichia coli, obtain recombination bacillus coli；
S2, the recombination bacillus coli production isoeugenol monooxygenase activity aggregation using structure：
The recombination bacillus coli of structure is subjected to culture optimization, induction, the thalline after induction is collected by centrifugation, smudge cells with
Crude enzyme liquid is obtained, gained crude enzyme liquid is centrifuged and/or filtered, it is isoeugenol monooxygenase activity aggregation to obtain precipitation.
2. the construction method of isoeugenol monooxygenase activity aggregation according to claim 1, it is characterised in that step
S1 comprises the following steps：
S1-1, by bacterial strain E.coli BL21（DE3）IEM and E.coli BL21（DE3）PET30a-LipA-18A is cultivated,
E. coli BL21 is obtained respectively（DE3）IEM and E. coli BL21（DE3）pET30a-LipA-18A
Cell；
S1-2, the gene order according to isoeugenol monooxygenase, design four pairs of PCR primers, including sense primer A, downstream draw
Thing B1, anti-sense primer B2, anti-sense primer B3 and anti-sense primer B4；Wherein sense primer A and anti-sense primer B4 is comprising digestion position
The specific primer of point；
S1-3, in sequence respectively using four pairs of PCR primers, with E. coli BL21（DE3）IEM is first time PCR
Template enters performing PCR amplification, and obtained PCR primer takes 1 μ L as the template of PCR next time after diluting 1000 times；Next time
PCR is expanded using last PCR primer as template, finally gives PCR primer target gene isoeugenol monooxygenase, life
Entitled IEM-1；
S1-4, the target gene IEM-1 is purified；
S1-5, from E. coli BL21（DE3）Plasmid pET30a-LipA-18A is extracted in pET30a-LipA-18A；
S1-6, double digestion is carried out to the target gene IEM-1 and plasmid pET30a-LipA-18A respectively；
S1-7, the double digestion product progress nucleic acid electrophoresis by target gene IEM-1 and plasmid pET30a-LipA-18A, reclaim double
Digestion products；
S1-8, the product after target gene IEM-1 and plasmid pET30a-LipA-18A double digestion recyclings is attached, obtained
To the active aggregations of connection product isoeugenol monooxygenase -18A, pET30a-IEM-18A is named as；
S1-9, with the connection product pET30a-IEM-18A convert E. coli BL21 (DE3), obtain recombinant bacterium
E.coli BL21(DE3) pET30a-IEM-18A。
3. the construction method of isoeugenol monooxygenase activity aggregation according to claim 2, it is characterised in that step
In S1-2：
Sense primer A sequence is as follows：5’-tagctacatatgatggcaacgtttaccgcaatgatc-3’
Anti-sense primer B1 sequence is as follows：5’-gcggccgccttatctctcgaggttcttagactgccaac-3’
Anti-sense primer B2 sequence is as follows：5’-gtgcaacgctgctcggagacggaagggtagctt-3’
Anti-sense primer B3 sequence is as follows：5’-gactgccaacaaccgtgcaacgctgctcggaga-3’
Anti-sense primer B4 sequence is as follows：5’-tagctaaagctttcagttcttagactgccaacaaccgtgc-3’.
4. the construction method of isoeugenol monooxygenase activity aggregation according to claim 2, it is characterised in that step
S2 comprises the following steps：
S2-1, the recombination bacillus coli of structure is inoculated in received containing card mycin liquid LB medium in cultivate to be planted
Sub- liquid；
S2-2, take the seed liquor of acquisition be inoculated in received containing card mycin solid-state LB culture mediums in cultivate；
S2-3, in the solid-state LB culture mediums add derivant induced；
The thalline after induction is collected by centrifugation in S2-4, low-temperature and high-speed, and thalline is resuspended with Glycine-NaOH cushioning liquid, is made into
Cell suspension, smudge cells；
S2-5, centrifugation, add isometric Glycine-NaOH or sodium carbonate-sodium hydroxide buffer solution in sediment,
Soft to be resuspended, the crude enzyme liquid of acquisition is centrifuged and/or filtered, and it is isoeugenol monooxygenase activity aggregation to obtain precipitation.
5. the construction method of isoeugenol monooxygenase activity aggregation according to claim 1, it is characterised in that the structure
Construction method is further comprising the steps of：
S3, isoeugenol monooxygenase activity aggregation immobilization：
By cross-linked enzyme aggregate technology, using glutaraldehyde as cross linker, the isoeugenol monooxygenase that step S2 is obtained is lived
Property aggregation carry out carrier-free immobilization.
6. the construction method of isoeugenol monooxygenase activity aggregation according to claim 5, it is characterised in that step
S3 comprises the following steps：
Glutaraldehyde solution is added dropwise in S3-1, the crude enzyme liquid obtained at low temperature toward step S2, stirs；
S3-2, at low temperature high speed centrifugation, the sediment of acquisition are the isoeugenol monooxygenase activity aggregation of immobilization, i.e.,
Immobilised enzymes；
S3-3, by sodium carbonate-sodium hydroxide buffer solution to immobilised enzymes be resuspended, cleaning immobilised enzymes surface attachment penta 2
Aldehyde；
S3-4, low-temperature and high-speed centrifugation, immobilised enzymes is resuspended using sodium carbonate-sodium hydroxide buffer solution.
7. a kind of method that isoeugenol monooxygenase activity aggregation conversion isoeugenol produces vanillic aldehyde, it is characterised in that
Comprise the following steps：
T1, build recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation：
Small peptide 18A with amphipathic self-assembling function is connected with target gene isoeugenol monooxygenase, isobutyl is built fragrant
Phenol monooxygenase -18A activity aggregations, and in expression in escherichia coli, obtain recombination bacillus coli；
T2, the recombination bacillus coli conversion isoeugenol production vanillic aldehyde using structure：
The recombination bacillus coli of structure is subjected to culture optimization, induction, the thalline after induction is collected by centrifugation, thalline is resuspended and matches somebody with somebody
Into cell suspension, add isoeugenol and converted, obtain the reaction solution containing vanillic aldehyde.
8. isoeugenol monooxygenase activity aggregation conversion isoeugenol according to claim 7 produces the side of vanillic aldehyde
Method, it is characterised in that step S1 comprises the following steps：
T1-1, by bacterial strain E.coli BL21（DE3）IEM and E.coli BL21（DE3）PET30a-LipA-18A is cultivated,
E. coli BL21 is obtained respectively（DE3）IEM and E. coli BL21（DE3）pET30a-LipA-18A
Cell；
T1-2, the gene order according to isoeugenol monooxygenase, design four pairs of PCR primers, including sense primer A, downstream draw
Thing B1, anti-sense primer B2, anti-sense primer B3 and anti-sense primer B4；Wherein sense primer A and anti-sense primer B4 is comprising digestion position
The specific primer of point；
T1-3, in sequence respectively using four pairs of PCR primers, with E. coli BL21（DE3）IEM is first time PCR
Template enters performing PCR amplification, and obtained PCR primer takes 1 μ L as the template of PCR next time after diluting 1000 times；Next time
PCR is expanded using last PCR primer as template, finally gives PCR primer target gene isoeugenol monooxygenase, life
Entitled IEM-1；
T1-4, the target gene IEM-1 is purified；
T1-5, from E. coli BL21（DE3）Plasmid pET30a-LipA-18A is extracted in pET30a-LipA-18A；
T1-6, double digestion is carried out to the target gene IEM-1 and plasmid pET30a-LipA-18A respectively；
T1-7, the double digestion product progress nucleic acid electrophoresis by target gene IEM-1 and plasmid pET30a-LipA-18A, reclaim double
Digestion products；
T1-8, the product after target gene IEM-1 and plasmid pET30a-LipA-18A double digestion recyclings is attached, obtained
To the active aggregations of connection product isoeugenol monooxygenase -18A, pET30a-IEM-18A is named as；
T1-9, with the connection product pET30a-IEM-18A E. coli BL21 (DE3) is transferred to, obtains recombinant bacterium
E.coli BL21(DE3) pET30a-IEM-18A。
9. isoeugenol monooxygenase activity aggregation conversion isoeugenol according to claim 8 produces the side of vanillic aldehyde
Method, it is characterised in that in step T1-2：
Sense primer A sequence is as follows：5’-tagctacatatgatggcaacgtttaccgcaatgatc-3’
Anti-sense primer B1 sequence is as follows：5’-gcggccgccttatctctcgaggttcttagactgccaac-3’
Anti-sense primer B2 sequence is as follows：5’-gtgcaacgctgctcggagacggaagggtagctt-3’
Anti-sense primer B3 sequence is as follows：5’-gactgccaacaaccgtgcaacgctgctcggaga-3’
Anti-sense primer B4 sequence is as follows：5’-tagctaaagctttcagttcttagactgccaacaaccgtgc-3’.
10. isoeugenol monooxygenase activity aggregation conversion isoeugenol production vanillic aldehyde according to claim 8
Method, it is characterised in that step T2 comprises the following steps：
T2-1, the recombination bacillus coli of structure is inoculated in received containing card mycin seed culture medium in cultivate to obtain seed
Liquid；
T2-2, obtain seed liquor be inoculated in received containing card mycin fermentation medium in culture；
T2-3, in the fermentation medium add derivant induced；
T2-4, by the zymotic fluid low-temperature centrifugation after induction, collect thalline, thalline be resuspended with Glycine-NaOH cushioning liquid,
It is made into cell suspension；
T2-5, take cell suspension, add isoeugenol and converted, reaction solution of the acquisition containing vanillic aldehyde.
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| CN114072507A (en) * | 2019-04-29 | 2022-02-18 | 科纳根公司 | Biosynthesis of vanillin from isoeugenol |
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