CN101397568A - Bacteria ferulic acid decarboxylase gene and crystal structure thereof - Google Patents
Bacteria ferulic acid decarboxylase gene and crystal structure thereof Download PDFInfo
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Abstract
The invention relates to an expression, purification, crystallization method, structure elucidation and the field of application technology of forulic acid decarboxylase, which belongs to the field of molecular biology and microbiology application. The invention obtains the coding area sequence of the key enzyme forulic acid decarboxylase which decomposes forulic acid to generate 4-vinyl guaiacol in Enterobacter sp.Px6-4 by the gene clone method, expresses the enzyme in a large amount in the colon bacillus ( E.coli BL21 ) by the heterogenous expressing technology, obtains the forulic acid decarboxylase pure products by the purification method and the forulic acid decarboxylase protein crystallization by the hanging-drop crystallization process, and grasps the structure and active centre of the enzyme. The invention can obtain a large amount of highly active forulic acid decarboxylase, and provide theoretical guidance for reconstructing engineering bacteria and improving the output of 4-vinyl guaiacol by analyzing the active centre of the enzyme, thus having good potential application.
Description
Technical field:
The present invention relates to a kind of bacteria ferulic acid decarboxylase gene and crystalline structure thereof, belong to molecular biology and applied microbiology technical field.
Background technology:
Forulic acid (4-hydroxyl-3 methoxyl groups-2-vinylformic acid) is a kind of botanic hydroxycinnamic acid that extensively exists, and is the highest phenolic acid of content in the wheat bran, can reach its dry weight 0.5～1%.It is mainly crosslinked by ester bond and polysaccharide and xylogen, or self esterification or etherificate form two forulic acid.Many plants, fungi, bacterium, actinomycetes, little algae etc. can be decomposed forulic acid the generation vanillin food grade,1000.000000ine mesh, forulic acid is produced the precursor substance of vanillin food grade,1000.000000ine mesh as bio-transformation, have be easy to get, inexpensive and to advantages such as the toxicity of microorganism are less, therefore to being that the research that precursor substance transforms the pathways metabolism that generates vanillin food grade,1000.000000ine mesh has very big theory significance and productive value with the forulic acid.
Vanillin food grade,1000.000000ine mesh (4-hydroxy 3-methoxybenzene formaldehyde), have another name called Vanillin, be one of a kind of important broad spectrum type spices, fragrance is quiet and tastefully laid out, frank, industries such as makeup, cigarette, cake, candy and bake be can directly apply to, plant growth promoter, sterilant, lubricating oil antifoams etc. also can be used as.Vanillin food grade,1000.000000ine mesh is important organic synthesis intermediate, and vanillin food grade,1000.000000ine mesh is mainly used in foodstuff additive at home, is constantly widened in the application of field of medicaments in recent years, has become the Application Areas that vanillin food grade,1000.000000ine mesh has development potentiality most.Remove in addition, it also can be used as lustering agent in electroplating industry, be used as ripener etc. in the agricultural.
Generate in the approach of vanillin food grade,1000.000000ine mesh in the forulic acid metabolism, ferulic acid decarboxylase catalysis forulic acid generates 4-ethyl guaiacol, is the key enzyme that the forulic acid metabolism forms vanillin food grade,1000.000000ine mesh.The 4-vinyl guaiacol can produce a kind of fragrance of special aldehydes matter as the extremely low material of a kind of threshold of feelings in addition.This material can improve the organoleptic quality of food, and to wine, food, the natural fragrance of beverage also has a significant impact.
In the past to being that the research that substrate conversion forms vanillin food grade,1000.000000ine mesh mainly concentrates on the pathways metabolism aspect with the forulic acid, some main pathways metabolisms being in the news gradually at present.Yet the gene that participates in the decarboxylase of bacterium Enterobacter sp.Px6-4 catalysis forulic acid formation 4-ethyl guaiacol also is not in the news.For the structure of ferulic acid decarboxylase and the relation between the function among the more deep understanding Enterobacter sp.Px6-4, the present invention has obtained the crystal of ferulic acid decarboxylase by gene clone, expression and hanging drop diffused junction crystal method, and by the X-diffraction method crystalline structure of ferulic acid decarboxylase is resolved.By literature search, find open report with content same document of the present invention.
Summary of the invention:
The present invention clone from the bacterium Enterobacter sp.Px6-4 that can transform forulic acid formation 4-ethyl guaiacol and vanillin food grade,1000.000000ine mesh obtains the encoding gene of ferulic acid decarboxylase.This bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China, BeiJing ZhongGuanCun; Preservation date: on April 12nd, 2007; Preserving number: CGMCC No.1999.
The objective of the invention is to obtain the encoding gene of bacterium Enterobacter sp.Px6-4 ferulic acid decarboxylase by the method amplification of gene clone, in intestinal bacteria E.coli, express, and utilization hanging drop diffusion process obtains the crystallization of protein of ferulic acid decarboxylase, and by the X-diffraction method structure and the active centre of this enzyme resolved.For the relation between the 26S Proteasome Structure and Function of further research ferulic acid decarboxylase and change 4-vinyl guaiacol output theoretic guidance is provided.
1. from bacterium Enterobacter sp.Px6-4, clone the encoding gene that obtains ferulic acid decarboxylase among the present invention, and the method for successful expression comprises the steps: in intestinal bacteria E.coli
1) the present invention is according to GenBank (international nucleotide sequence database, http://www.ncbi.nlm.nih.gov/) gene order (AF017117 of the hydroxycinnamic acid decarboxylase of 6 bacterial origins upward reporting, AJ276891, AJ27863, AJ492219, U63827 and X84815) designed pair of degenerate primers PS and Px2 (table 1) be used to the to increase conserved sequence of ferulic acid decarboxylase.
2) designed 6 Auele Specific Primer PxT31 according to this conservative fragments, PxT32, PxT33, PxT51, PxT52, PxT53 (table 1), and use DNA Walking SpeedUp
TMThe amplification of Premix test kit obtains the unknown nucleotide sequence at Enterobacter sp.Px6-4 ferulic acid decarboxylase conserved sequence two ends.
3) splice the total length coding gene sequence (seeing ferulic acid decarboxylase gene order table for details) that obtains Enterobacter sp.Px6-4 ferulic acid decarboxylase by the DNAman biological software.
4) designed specificity Px1e and Px2e (table 1) the Enterobacter sp.Px6-4 ferulic acid decarboxylase encoding sequence that is used to increase according to the total length coding gene sequence of Enterobacter sp.Px6-4 ferulic acid decarboxylase.
The used primer sequence of table 1. the present invention
5) the PCR product is carried out double digestion with EcoR I and Hind III, the small segment after reclaiming purifying enzyme and cutting also is connected construction of expression vector with pET-28a behind EcoR I and Hind III double digestion.
6) with the expression vector Transformed E .coli BL21 that builds; After IPTG induces, the activated ferulic acid decarboxylase of E.coli BL21 great expression.
7) through behind affinity column, anion column, the drainage column purifying, obtain the pure enzyme of ferulic acid decarboxylase.
2. definite method in the crystalline structure of ferulic acid decarboxylase and active centre among the present invention:
1) ferulic acid decarboxylase of purifying is concentrated into 10mg/mL after, in 0.1M HEPES (pH7.3), 26%w/v PEG10,000 crystallization condition obtains the crystallization of ferulic acid decarboxylase down by the hanging drop crystallization process.
2) ferulic acid decarboxylase of purifying being concentrated into 10mg/mL mixes with the Sodium Ferulate of 7mM, in 0.1M HEPES (pH7.3), 26%w/v PEG10,000 crystallization condition obtains the crystal of ferulic acid decarboxylase and Sodium Ferulate complex body down by the hanging drop crystallization process.
3) collect and analytical procedure 1) in the ferulic acid decarboxylase X-ray diffraction in crystals data that obtain, obtain the electron density of ferulic acid decarboxylase.
4) collect and analytical procedure 2) in the X-ray diffraction in crystals data of the complex body that obtains, acquisition and ferulic acid decarboxylase and Sodium Ferulate bonded electron density.
5) based on the electron density of the ferulic acid decarboxylase that obtains in the step 3), obtain the ferulic acid decarboxylase fine three dimensional structure.
6) ferulic acid decarboxylase and the Sodium Ferulate bonded electron density to obtain in the step 4) obtains the fine three dimensional structure of ferulic acid decarboxylase and Sodium Ferulate complex body, thereby determined the active centre of ferulic acid decarboxylase
Step 3) and 4) the X-ray diffraction in crystals data gathering can be adopted following method with arrangement in:
At first use the nylon crystal rings of Hampton Research company, from the crystal soak solution, obtain the crystal that soaks certain hour (soak time was controlled between 2-48 hours), and use the cooling system of Rigaku company or the cooling system of Oxford Cyrosystem company rapidly, crystal is refrigerated to subzero 150 ℃-180 ℃ in the cryogenic nitrogen air-flow that above-described two kinds of refrigeration systems produce; Make X ray pass through crystal, use the precession method to collect X ray diffracting data.
After collecting the X-ray diffraction in crystals data, carry out corresponding data processing according to following step:
At first, use HKL2000 etc. (to comprise Mosflm, D
*Trek etc.) method of the data processing that diffraction data handling procedure bag etc. is commonly used is handled the diffraction data that collection obtains, and obtains complete data file.
Secondly, use the data processing methods commonly used known in the art such as various molecular replacements such as Phaser, Molrep (MolecularReplacement) program in CNS or the CCP4 routine package, utilize the method for molecular replacement (MolecularReplacement), (PDB code:2P8G) is initial model with phenolic acid decarboxylase crystalline structure, obtains the fine three dimensional structure of ferulic acid decarboxylase; Parent crystalline structure with ferulic acid decarboxylase is an initial model, obtains the fine three dimensional structure of ferulic acid decarboxylase and Sodium Ferulate complex body.
The present invention finds being characterized as of overall structure of ferulic acid decarboxylase: the resolving power of the monomer structure of ferulic acid decarboxylase is
Cylindrical, overall size is
Constitute by 9 strands of βZhe Die thighs, 2 α spirals and 3 sections η helical conformations; Each monomer molecule is divided into three parts again promptly: centre portions, spiral helicine bottom and C-terminal extend; Wherein, centre portions is from βZhe Die thigh 1 to βZhe Die thigh 8 with βZhe Die thigh 9, and these nine strands antiparallel βZhe Die crural rings are around having formed open-ended β barrel-like structure; The spirrillum bottom is made of alpha helical conformation 1, alpha helical conformation 2 and η helical conformation 1, η helical conformation 2; It is to be positioned at βZhe Die thigh 9 segment length afterwards to curl that C-terminal extends, and wherein η helical conformation 2 is positioned at curling middle part.The present invention finds that ferulic acid decarboxylase substrate binding site (active centre) is characterized by: the substrate analogue Sodium Ferulate is attached to the middle part and the spiral helicine bottom at ferulic acid decarboxylase β barrel-like structure center; In the bottom of its semi-open type, the residue that βZhe Die thigh 8, βZhe Die thigh 9 and the hairpin structure between βZhe Die thigh 1 and βZhe Die thigh 2, βZhe Die thigh 3 and βZhe Die thigh 4 constitute has formed a hydrophobic relatively substrate binding pocket; Hairpin structure between βZhe Die thigh 1 and βZhe Die thigh 2, βZhe Die thigh 3 and βZhe Die thigh 4 has been taken on the effects of two fan door-plates in the front of substrate binding pocket, we at this with its called after door-plate 1 and door-plate 2; Be positioned at the 25th tryptophane and taken on the function of door lock; Under substrate binding pocket closing state, door-plate 1 and door-plate 2 move about 5 towards βZhe Die thigh 9
Pocket is closed and substrate and catalytic center are separated; Under the state that the substrate binding pocket is opened, door-plate 1 door-plate 2 is removed pocket is become open form from the form of closing; Under opened state, be formed centrally one 8 * 8 * 15 in the substrate combination
Pocket, help substrate and enter the active centre.
The present invention provide specific embodiments of the invention in order to explain in further detail below in conjunction with accompanying drawing.Following embodiment is only used for explaining the present invention, and not should be understood to limit by any way the scope of the invention.
Description of drawings:
Fig. 1. be expression and the purifying electrophoretogram of ferulic acid decarboxylase at intestinal bacteria E.coli.M: protein molecular weight standard; Lane 1: no IPTG inductive gross protein; Lane 2:IPTG inductive gross protein; Lane 3: with the protein of Ni-chelating column wash-out; Lane 4: with the protein of Resource Q column wash-out; Lane 5: with the ferulic acid decarboxylase of HiLoad 16/60 Superdex column purifying.
Fig. 2. the monomer structure of ferulic acid decarboxylase.The monomer structure of ferulic acid decarboxylase is a barrel-like structure, and size is
Fig. 3. be the crystalline structure figure of ferulic acid decarboxylase and Sodium Ferulate mixture.A. the substrate binding site of ferulic acid decarboxylase; B. the interactional amino acid molecular of Sodium Ferulate and ferulic acid decarboxylase.
Embodiment:
1. the extraction of bacterial genomes DNA
1) Enterobacter sp.Px6-4 is inoculated in liquid LB substratum, 37 ℃, 200rpm were cultivated 12 hours;
2) get centrifugal 2 minutes of the culture 12000rpm of 1mL, remove supernatant;
3) precipitation TE damping fluid (10mM Tris-HCl, pH8.0; 0.1mM EDTA pH8.0) washes twice;
4) add 30 μ L SDS (10%) and 15 μ L Proteinase Ks (20mg/mL) with the resuspended back of the TE damping fluid of 567 μ L, bathed 1 hour in 37 ℃ of temperature the back that is mixed;
5) add the CTAB/NaCl (10%CTAB, 0.7M NaCl) of 100 μ L NaCl (5M), 80 μ L successively, bathed 10 minutes in 65 ℃ of temperature the back that is mixed;
6) phenol of adding 700 μ L: chloroform: 5000rpm was centrifugal 5 minutes after primary isoamyl alcohol (25:24:1v/v) was put upside down and is mixed for several times;
7) supernatant is moved in the new EP pipe Virahol that adds 0.6 times of volume, centrifugal 10 minutes of 4 ℃, 12000rpm;
8) precipitation washes twice with 70% ethanol, and vacuum-drying is after 5 minutes, with the deionized water of 50 μ L with resolution of precipitate.
2.Enterobacter the acquisition of sp.Px6-4 ferulic acid decarboxylase conserved sequence
1) pcr amplification system
10?x?Pfu?Buffer 5μL
dNTP?Mixture 1μL
PS(10μM) 1μL
P?x?2(10μM) 1μL
Template <1μg
Pfu?DNA?Polymerase(5u/μL)?0.5μL
dH
2O 50μL
2) PCR response procedures
1 circulation of 95 ℃ of 5.5min
94℃ 1min
56℃ 40sec
35 circulations of 72 ℃ of 2min
1 circulation of 72 ℃ of 10min
1 circulation of 4 ℃ of ∞
3) purifying of Enterobacter sp.P x 6-4 ferulic acid decarboxylase conserved sequence reclaims: method sees that hundred Tyke biotech firm glue reclaim the test kit specification sheets
4) Enterobacter sp.P x 6-4 ferulic acid decarboxylase conserved sequence is connected with carrier pMD18-T
Linked system
SolutionI 5μL
The purpose fragment 4 μ L that reclaim
16 ℃ of water-baths of pMD18-T vector 1 μ L connection of spending the night
5) recombinant plasmid transformed E.coli DH5 α
A. taking out 100 μ L competent cell suspensions from-70 ℃ of refrigerators is placed on ice and thaws;
B. add and connect product, placed on ice 30 minutes; Thermal shock is 90 seconds in 42 ℃ of water-baths, and speed placed cooled on ice 3-5 minute behind the thermal shock;
C. in pipe, add 0.8-1mL LB liquid nutrient medium, back 37 shaking culture that are mixed 1 hour;
D. bacterium liquid 8000rpm is centrifugal 1 minute, abandons most of supernatant, stays about 100 μ L bacterium liquid to coat on the Amp screening flat board, faces up to place after 30 minutes 37 ℃ and be inverted and cultivated 10-20 hour.
6) the sub-nutrient solution of the segmental clone of purpose send the order-checking of order-checking company
3.Enterobacter the acquisition of sp.Px6-4 ferulic acid decarboxylase full length gene sequence
1) uses DNA Walking SpeedUp
TMThe amplification of Premix test kit obtains the unknown nucleotide sequence at ferulic acid decarboxylase conserved sequence two ends, and concrete grammar is referring to the specification sheets of test kit.
2) recovery of amplified production, connection and order-checking, method is the same.
3) Enterobacter sp.P x 6-4 ferulic acid decarboxylase full length sequence: the full length sequence that utilizes the DNAman biological software to splice to obtain ferulic acid decarboxylase (concrete sequence sees Appendix 1).
4.Enterobacter the amplification of sp.P x 6-4 ferulic acid decarboxylase coding region sequence
1) amplification of ferulic acid decarboxylase encoding sequence: utilize specificity P x 1e and P x 2e (table 1) amplification to obtain Enterobacter sp.P x 6-4 ferulic acid decarboxylase encoding sequence, concrete grammar is the same.
2) recovery of amplified production, connection and order-checking, method is the same.
5. make up the recombinant plasmid that has Enterobacter sp.P x 6-4 ferulic acid decarboxylase coding region sequence
1) enzyme of ferulic acid decarboxylase encoding sequence is cut processing, utilizes restriction enzyme EcoR I, Hind III enzyme to cut and handles pcr amplification product and carrier pET-28a.
Enzyme is cut system
EcoR?I 2.5μL
Hind?III 2.5μL
Coding region sequence/pET-28a 30 μ L
dd?H
2O 15μL
37 ℃ of temperature of 10 x Buffer M, 5 μ L were bathed 6 hours
2) Enterobacter sp.P x 6-4 ferulic acid decarboxylase encoding sequence and pET-28a's after enzyme is cut processing is connected.
Linked system
pET-28a 2μL
3) conversion of recombinant plasmid and screening: method is the same.
6. the abduction delivering of ferulic acid decarboxylase in E coli BL21
1) has the recombinant plasmid transformed E.coliBL21 of Enterobacter sp.P x 6-4 ferulic acid decarboxylase coding region sequence.
A. taking out 100 μ L competent cell suspensions from-70 ℃ of refrigerators is placed on ice and thaws;
B. add and connect product, placed on ice 30 minutes; Thermal shock is 90 seconds in 42 ℃ of water-baths, and speed placed cooled on ice 3-5 minute behind the thermal shock;
C. add 0.8-1mL LB liquid nutrient medium in pipe, back 37 ℃ of shaking culture 1h are mixed; Centrifugal 1 minute of bacterium liquid 8000rpm abandons most of supernatant, stays about 100 μ L bacterium liquid to coat on the Amp screening flat board, faces up to place after 30 minutes 37 ℃ and be inverted and cultivated 10-20 hour.
2) IPTG induces the ferulic acid decarboxylase heterogenous expression
The a.LB flat board activated spawn of spending the night; 37 ℃ of LB liquid activation, 180rpm cultivated 3 hours;
B. cultivating behind the IPTG that adds 0.2mM after 3 hours 30 ℃, 200rpm by LB37 ℃ of liquid of 1% inoculum size switching, 180rpm spends the night and induces.
7. the purifying of ferulic acid decarboxylase
1) activated seed
A. with the E.coli BL21 that has recombinant plasmid as seed;
B. the dull and stereotyped activatory seed of going up is transferred in 50mL liquid LB substratum, and 37 ℃, 200rpm shook the liquid LB substratum of the inoculum size switching 1L by 3% 3 hours, 37 ℃, 200rpm shook about 2 hours, and adding the IPTG final concentration is 0.2mM, 30 ℃, 200rpm spends the night and induces;
C.8000rpm collected thalline in centrifugal 20 minutes,, wash twice with the Tris-Hcl damping fluid (pH7.0) of 50mM;
D. resuspended with the 1L fermented liquid with the ratio of the 50mM of 30mL, Tris-Hcl damping fluid (pH7.0).
2) ultrasonic disruption obtains crude enzyme liquid
The resuspended liquid of cell is positioned on ice, and the frequency with 50% is stopped 12 seconds about 20 minutes of condition ultrasonication, till limpid fully after ultrasonic 8 seconds.
3) crude enzyme liquid is crossed affinity column
A. select the nickel post as affinity column;
D. the Tris-Hcl damping fluid (pH7.0) of using 50mM is gone up sample after with the pillar balance;
C. continue position and pillar bonded impurity to be washed away with the Tris-Hcl damping fluid (pH7.0) of 50mM;
D. use Tris-Hcl damping fluid (pH7.0)+100mM imidazoles wash-out of 50mM, collect elutriant.
4) the enzyme liquid dialysis treatment of wash-out
The enzyme liquid of wash-out is packed in the dialysis tubing, as dialyzate, be positioned on ice dialysis treatment 24 hours with the Tris-Hcl damping fluid (pH7.0) of 20mM.
5) the enzyme liquid after the dialysis is crossed anion column
A. the Tris-Hcl damping fluid (pH7.0) of using 20mM is gone up sample after with the pillar balance;
D. continue position and pillar bonded impurity to be washed away with the Tris-Hcl damping fluid (pH7.0) of 20mM;
C. continue to make gradient elution, collect the elutriant that contains target protein with the Tris-Hcl damping fluid (pH7.0) of damping fluid 1M NaCl+20mM.
6) elutriant that contains target protein is crossed drainage column
A. will cross the ammonium sulfate that adds 0.5M in the elutriant that contains target protein behind the anion column;
D. the Tris-Hcl damping fluid (pH7.0) that adds 50mM with the ammonium sulfate of 0.5M is gone up sample after with the pillar balance;
C. continuing the Tris-Hcl damping fluid (pH7.0) that ammonium sulfate with 0.5M adds 50mM washes away position and pillar bonded impurity;
D. after using the Tris-Hcl damping fluid (pH7.0) of 50mM to make gradient elution, collect the elutriant that contains target protein with the deionized water wash-out.
7) purity of SDS electrophoretic analysis ferulic acid decarboxylase, experimental result is seen accompanying drawing 1.
8. ferulic acid decarboxylase crystallization and the structural analysis behind the purifying
1) ferulic acid decarboxylase crystallization
A. the ferulic acid decarboxylase behind the purifying is concentrated to 10mg/mL;
B. in the syringe of 10mL, insert silicone oil;
C. on each culture hole limit, draw the silicone oil circle that 2mm is wide;
D. 0.1M HEPES (the pH7.3)+26%w/v PEG10 that in the culture hole of tissue culturing plate, adds 400 μ L, 000 damping fluid;
C. cover glass is placed on the clean filter paper, only contacts its edge;
D. on cover glass, select 0.5 μ L damping fluid and 0.5 μ L protein soln with sample injector;
E. clamp the cover glass edge with tweezers,, drop is dangled under cover glass its upset;
F. put cover glass on the corresponding hole of culture plate, guarantee that the cover glass edge is airtight with the hole;
G. with pointing with even and strong being pressed together on the hole of cover glass;
H. soft cover lid places 16 ℃ of storages.
2) ferulic acid decarboxylase and Sodium Ferulate cocrystallization
A. the ferulic acid decarboxylase behind the purifying is concentrated to 10mg/mL;
B. in the syringe of 10mL, insert silicone oil;
C. on each culture hole limit, draw the silicone oil circle that 2mm is wide;
D. 0.1M HEPES (the pH7.3)+26%w/v PEG10 that in the culture hole of tissue culturing plate, adds 400 μ L, 000 damping fluid;
C. cover glass is placed on the clean filter paper, only contacts its edge;
D. on cover glass, select 7mM Sodium Ferulate, the 0.5 μ L protein soln of 0.5 μ L damping fluid, 0.5 μ L with sample injector;
E. clamp the cover glass edge with tweezers,, drop is dangled under cover glass its upset;
F. put cover glass on the corresponding hole of culture plate, guarantee that the cover glass edge is airtight with the hole;
G. with pointing with even and strong being pressed together on the hole of cover glass;
H. soft cover lid places 16 ℃ of storages.
3) collection of ferulic acid decarboxylase crystalline structure data and structure elucidation
By the crystal of hanging drop diffusion process acquisition ferulic acid decarboxylase, ferulic acid decarboxylase precursor structure data utilize Synchrotron Radiation Mar165 ccd detector to collect data at the cold condition of 100K.Diffraction data is used the HKL2000 software package and is handled.(PDB is numbered the crystalline structure of ferulic acid decarboxylase: 2P8G) use the PHASER program as search model and obtain initial phase by molecular replacement with the crystalline structure of phenolic acid decarboxylase.The ferulic acid decarboxylase crystallization belongs to P2
1Spatial group comprises two monomer molecules in structure cell asymmetric cell (unit).
4) collection and the structure elucidation of the composite crystalline structure data of ferulic acid decarboxylase and Sodium Ferulate
The structure of ferulic acid decarboxylase and Sodium Ferulate compound crystal, is used the PHASER program and is obtained initial phase by molecular replacement as search model with the precursor structure of ferulic acid decarboxylase.The combination of Sodium Ferulate and decarboxylase makes up with reference to the 1Fo-Fc electron density map.And application software COOT and Refmac carry out final craft according to 2Fo-Fc and 1Fo-Fc electron density map to structure and rebuild and revise.Pass through R in position correction subsequently, not strict (restraints were relaxed) and the correction of volume of solvent of constraint
FreeGuidance realize.The correction PROCHECK program that model geometric is learned.The basis on location of solvent molecule is in the stereochemical rational peak value on σ A-weighted Fo-Fc difference electron density map.The diffraction data of ferulic acid decarboxylase crystalline structure and compound crystal structure sees Table 2.Ferulic acid decarboxylase and Sodium Ferulate composite crystalline structure are seen accompanying drawing 2.
The collection and the correction of table 2. crystalline diffraction data
aR
Merge=∑
h∑
1| I
Ih-＜I
h|/∑
h∑
I＜I
h,＜I
hBe the average I of observed value
IhWith reference to h
bR
Work=∑ (|| F
p(obs) |-| F
p(calc) ‖)/and ∑ | F
p(obs) |; R
FreeThe point diffraction that refers to not to be included in preferential refinement extracts the R factor after the subclass (5%)
cThe numerical value of the corresponding highest resolution of value representation in the parenthesis.
9. the application of ferulic acid decarboxylase
The ferulic acid decarboxylase of clone of the present invention, expression, purifying can decompose forulic acid fast and efficiently makes it to generate its decarboxylate 4-vinyl guaiacol.The product that obtains by the enzyme effect meets the requirement of the European Economic Community (EEC) to natural product.
The present invention has resolved the three-dimensional structure of ferulic acid decarboxylase by the X-ray diffraction method, and the substrate binding site and the active centre of this enzyme have been determined by diffraction result to the cocrystallization of ferulic acid decarboxylase and Sodium Ferulate, conformation between the amino acid of omnibearing this enzyme of reflection of data energy on these crystallologies, provide theoretic guidance for the structure of modification of ferulic acid decarboxylase improves 4-vinyl guaiacol output, have a good application prospect.
Among the related herein various experimental articles (including but not limited to: chemical reagent, biological products, cell, organism, instrument etc.), special or be difficult for obtaining for those, Wen Zhongjun has indicated manufacturers, reference or detailed preparation method; Without what specify, be the normal experiment articles for use, can pass through variety of way (for example buy, preparation etc.) voluntarily acquisition easily.
Ferulic acid decarboxylase gene [1] [1] .ST25
SEQUENCE?LISTING
＜110〉Yunnan University
＜120〉a kind of bacteria ferulic acid decarboxylase gene and crystalline structure thereof
<130>?01
<160>?1
<170>?PatentIn?version?3.1
<210>?1
<211>?930
<212>?DNA
<213>?Enterobacter?sp.Px6-4
<220>
<221>?fad?gene
<222>?(100)..(606)
<223>
<220>
<221>?promoter
<222>?(8)..(57)
<223>
<400>?1
Claims (3)
1, a kind of bacteria ferulic acid decarboxylase gene is characterized in that the nucleotide sequence of ferulic acid decarboxylase encoding gene is as follows:
aattaatcta?aaatttggat?gaatttgatt?caaacatagc?gttattcatt?cgttttttga 60
aggaataaga?tcgtctcatc?aaaacaaagg?agacatctta?tgaacacctt?cgacaaacat 120
gatttaagcg?gcttcgtcgg?caaacatctg?gtttatacct?acgataacgg?ctgggaatat 180
gaaatttacg?tcaaaaacga?aaacaccctc?gactaccgta?ttcacagcgg?cctggtcggc 240
aaccgctggg?tgaaagacca?gcaggcgtac?atcgtccgcg?ttggggagag?catctataaa 300
atctcctgga?ccgagcccac?cggcaccgac?gtgagcctga?ttgtgaacct?gggtgacagc 360
ctgttccacg?gcacgatctt?cttcccgcgc?tgggtaatga?acaatccgga?aaaaaccgtc 420
tgcttccaga?acgatcacat?tccgttgatg?aatagctatc?gcgatgcggg?cccggcatat 480
ccaaccgaag?tgattgatga?atttgccact?attacttttg?ttcgcgactg?cggtgccaac 540
aatgaaagcg?ttatcgcgtg?cgctgccagt?gaacttccaa?aaaactttcc?tgataattta 600
aaataagcgc?gacaattaac?taaaaagaaa?agttcccggc?ccatttcata?tttattcgaa 660
atggtcggga?atttttttgt?ttgttgatgt?ttggtttaga?taaaaagcgc?gttaacggtg 720
agcgtaatca?caaaacaaat?aattcataat?ttaataacgc?tttgattatt?aattggtttt 780
2, a kind of bacteria ferulic acid decarboxylase crystalline structure, it is characterized by: the resolving power of the monomer structure of ferulic acid decarboxylase is
Cylindrical, overall size is
Constitute by 9 strands of βZhe Die thighs, 2 α spirals and 3 sections η helical conformations; Each monomer molecule is divided into three parts again promptly: centre portions, spiral helicine bottom and C-terminal extend; Wherein, centre portions is from βZhe Die thigh 1 to βZhe Die thigh 8 with βZhe Die thigh 9, and these nine strands antiparallel βZhe Die crural rings are around having formed open-ended β barrel-like structure; The spirrillum bottom is made of alpha helical conformation 1, alpha helical conformation 2 and η helical conformation 1, η helical conformation 2; It is to be positioned at βZhe Die thigh 9 segment length afterwards to curl that C-terminal extends, and wherein η helical conformation 2 is positioned at curling middle part.
3, the described bacteria ferulic acid decarboxylase of claim 2 crystalline structure is characterized in that the substrate binding site of bacteria ferulic acid decarboxylase is: the substrate analogue Sodium Ferulate is attached to the middle part and the spiral helicine bottom at ferulic acid decarboxylase β barrel-like structure center; In the bottom of its semi-open type, the residue that βZhe Die thigh 8, βZhe Die thigh 9 and the hairpin structure between βZhe Die thigh 1 and βZhe Die thigh 2, βZhe Die thigh 3 and βZhe Die thigh 4 constitute has formed a hydrophobic relatively substrate binding pocket; Hairpin structure between βZhe Die thigh 1 and βZhe Die thigh 2, βZhe Die thigh 3 and βZhe Die thigh 4 has been taken on the effects of two fan door-plates in the front of substrate binding pocket, we at this with its called after door-plate 1 and door-plate 2; Be positioned at the 25th tryptophane and taken on the function of door lock; Under substrate binding pocket closing state, door-plate 1 and door-plate 2 move approximately towards βZhe Die thigh 9
Pocket is closed and substrate and catalytic center are separated; Under the state that the substrate binding pocket is opened, door-plate 1 door-plate 2 is removed pocket is become open form from the form of closing; Under opened state, be formed centrally one in the substrate combination
Pocket, help substrate and enter the active centre.
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