CN103911394A - Method of screening 1-deoxyxylulose-5-phosphate racemase inhibitor from marine microorganism fermentation broth - Google Patents

Method of screening 1-deoxyxylulose-5-phosphate racemase inhibitor from marine microorganism fermentation broth Download PDF

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CN103911394A
CN103911394A CN201410093680.6A CN201410093680A CN103911394A CN 103911394 A CN103911394 A CN 103911394A CN 201410093680 A CN201410093680 A CN 201410093680A CN 103911394 A CN103911394 A CN 103911394A
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dxr
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marine microorganism
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CN103911394B (en
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王蔚
李芊
杨慧
陈卓
胡彬
陈建明
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Minjiang University
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Third Institute of Oceanography SOA
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Abstract

The invention discloses a method of screening a 1-deoxyxylulose-5-phosphate racemase inhibitor from a marine microorganism fermentation broth. The method includes: 1) a step of preparing a marine microorganism fermentation broth sample, namely, a step of inoculating a fungus, fermenting, extracting metabolites, concentrating and eluting; 2) a step of designing a primer and performing PCR amplification by adopting the whole genome of vibrio vulnificus as a template, cloning a vector, converting escherichia coli, inducing expression of a target protein, and subjecting the target protein to elution, dialysis, ultrafiltration and desalting to obtain His-DXR; 3) a step of reacting a bacterial suspension, a resazurin solution and the marine microorganism fermentation broth sample obtained by the step 1), measuring the fluorescence value, calculating the inhibition rate through an equation, and screening the fraction the inhibition rate of which is higher than 80%; and 4) reacting the fraction screened in the step 3) and the His-DXR obtained in the step 2), calculating the inhibition rate, and screening the fraction the inhibition rate of which is not less than 50%. The method is simple and accurate, and lays a foundation for development of novel antibacterial medicines adopting Dxr as a therapeutic target or development of leading compounds.

Description

A kind of method of screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor from marine microorganism fermented liquid
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method of screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor from marine microorganism fermented liquid.
Background technology
Since nineteen twenty-nine Fu Laiming has found penicillin from fungi, microorganism active meta-bolites becomes the valuable source of drug discovery.The application identity of penicillin the beginning in microbiotic epoch, and increasing Lu Yuan microbial metabolites is developed to natural drug.In in the past more than 70 year, from microorganism, find about 30000-50000 kind natural product, wherein more than 10000, kind has biological activity, kind more than 8000 is antibacterial and antineoplastic agent, so far have according to incompletely statistics more than 100 kind by microorganisms producing and be applied to clinical agricultural chemicals, antineoplastic agent and microbiotic, annual global microbiotic output exceedes 100,000 tons, and the output value exceedes more than 50 hundred million (five strain thalassiomycetes secondary metabolite and bioactivity research thereof, 2010).But, to the studying for a long period of time in performance history of Lu Yuan microorganism, from Lu Sheng microbial secondary meta-bolites, find that new lead compound or antibiotic difficulty are increasing, and the exploitation that day by day seriously makes new drug of resistance problem is faced with severe challenge.
Ocean covers 71% of earth surface-area, is origin of life ground.In animal kingdom, 28 main animal doors have 26 to be to live in water, wherein also have 8 doors for completely aquatic, estimate that marine animal approximately has more than 50 ten thousand kinds.In marine organisms, marine microorganism is because it is lived in oligotrophic, weakly alkaline saliferous ocean environment, in order to maintain its vital movement, form physiological characteristic and the metabolic mechanisms such as unique resistance to famine, alkaline-resisting and salt tolerant, in metabolism and growth process, produced and be different from a large amount of novel structures of land microorganism, active strong secondary metabolite.In addition, because marine microorganism has that growth cycle is short, the restriction that is not subject to the aspect such as season and natural biology amount, the artificial advantage such as large scale culturing, natural be subject to increasing attention.With regard to finding new compound and microbiotic, only there is the separated evaluation of marine microorganism of 1% left and right at present, there is the great potential that produces novel bioactive material, people have expressed great hope for therefrom finding new specific medicament.
In recent years, along with the development of the subjects such as molecular biosciences, study biology, information biology, increasingly deep to the research of terpenoid, its biosynthetic pathway is also clear gradually, has been cloned and analyzed many key genes.Comprising one of relevant enzyme in Terpene biosynthesis approach: deoxy-D-xylulose sugar phosphoric acid salt reduction isomerase (DXR), DXR catalysis DXP generates MEP, is the rate-limiting enzyme of DXP approach.DXR is the target site of potential antimalarial agent, microbiotic and weedicide, and is a more efficient target site.Kuntz etc. (2005) report, fosmidomycin (fosmidomycin) is traditional DXR inhibitor, at 2 novel DXR inhibitors 4-(hydroxyamino)-4-oxobutylphosphonicacid and 4-[hydroxy (methyl) amino] in-4-oxobutylphosphonicacid, wherein the latter shows the activity of very high inhibition DXR.In bacterium, algae, plant and protozoon, found to exist DXR, but in human body, also do not found at present, therefore and antimalarial agent antibacterial with this target site development of DXR caused people's interest more.In the DXR gene of research Arabidopis thaliana, Rohdich etc. (2006) express the recombinant chou of removing after N end transit peptide sequence in intestinal bacteria (Escherichiacoli), the purifying protein obtaining by affinity chromatography can form MEP with DXP catalysis, catalytic rate is 5.6molmin-1mg-1 (albumen), this reaction needs NADPH to participate in, and needs divalent-metal ion Mn 2+and Mg 2+, optimum pH is 8.0, although the available NADH of NADPH substitutes, the speed of reaction after substituting is down to original 14%.
The mid-90 in 20th century, use molecular biology and comparative genomics method to confirm, have precursor substance isovaleryl tetra-sodium (IPP) or the dimethylallyl tetra-sodium (DMAPP) of a MEP (2-methyl-erythritol phosphoric acid) approach for the synthesis of isoprenoid at some bacterium, protozoon and plant plastid.This approach is different from mevalonic acid (MVA) approach in Mammals and plant endochylema completely.Due to the synthetic isoprenoid of this approach and derivative most important in biological metabolism and Cell wall synthesis, therefore MEP approach has become the focus of medicine target research, is one of medicine target of antibacterial, antimalarial extremely likely and weedicide.The approach of the synthetic isoprenoid material of tubercule bacillus is different from mammalian cell, depends on 2-C-methyl D-tetrahydroxybutane-4-phosphoric acid salt approach (MEP approach).Participate in the multiple bioprocess of tubercule bacillus through the synthetic isoprene compounds of MEP approach, MEP approach is essentially [to see EohH to the growth of tubercule bacillus, BrenanPJ, CrickDC.TheMycobacteriumtuberculosisMEP (2C-methyl-D-erythritol4-phosphate) pathwayasanewdrugtarget[J] .Tuberculosis2009,89 (1): 1-1].The 7 step reactions of MEP approach [4] experience, 7 kinds of enzyme catalysiss, change into isoprene bisphosphate (IPP) and dimethylallyl bisphosphate (DMAPP) by pyruvic acid and glyceraldehyde 3-phosphate.7 kinds of enzymes that participate in MEP approach are respectively: 1-deoxidation-D-5-lyxulose phosphate synthase (DXS), 1-deoxidation-D-5-lyxulose phosphate reductase enzyme (DXR), 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol synthetic enzyme (CMS), 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol kinase (CMK), 2C-methyl D-erythritol-2,4-encircles the third diphosphate synthase (MECDS), 1-hydroxy-2-methyl-2 (E)-butylene-4-diphosphate synthase (HMS) and 1-hydroxy-2-methyl-2 (E)-butylene-4-bisphosphate reductase enzyme (HMR).DXR is the highest enzyme of attention rate in MEP approach.First, DXR is the important enzyme of synthetic isoprene compounds; Secondly, it exists only in the bacteriums such as algae, plant, tubercule bacillus, in human body, does not express; Moreover, find that fosmidomycin (fosmidomycin, 7) is the effective anti-DXR medicine that enters clinical experimental stage.For these reasons, DXR is regarded as a good tuberculosis target spot and [sees SinghN, etal.Targetingthemethylerythritolphosphate (MEP) pathwayfornovelantimalarial, antibacterialandherbicidaldrugdiscovery:inhibitionof1-de oxy-D-xylulose-5-phosphatereductoisomerase (DXR) enzyme[J] .CurPharmDes, 207,13 (1): 1161-117].Researchist is take fosmidomycin as lead compound; successively find acetyl derivative (FR9098; 8); hydroxyurea analogue (compound 9); carboxylic acid analogue (compound 10); alpha-aromatic replaces analogue (compound 1) and [sees HaemersT; etal.Synthesisofalpha-sub-stitutedfosmidomycinanaloguesa shighlypotentPlasmodiumfalciparumgrowthinhibitors[J] .BiorgMedChemLet; the DXR inhibitor such as 2006,16 (7): 18-1891].
Summary of the invention
The object of the present invention is to provide a kind of easy, the method for screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor from marine microorganism fermented liquid.
For achieving the above object, the invention provides a kind of method of screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor from marine microorganism fermented liquid, it is characterized in that, comprise the steps,
1) preparation of marine microorganism fermentation broth sample: seawater PDA liquid nutrient medium inoculated fungi, after cultivation, continue fermentation as seed liquor, after fungi fermentation maturation, extract meta-bolites; And extract intracellular organic matter, the liquid concentration of extraction, to medicinal extract, obtains elementary medicinal extract;
Elementary medicinal extract utilizes normal phase silicagel column to carry out isocratic elution, and the time that goes out peak according to fignal center is collected elutriant, and concentrating under reduced pressure, obtains elementary cut;
Elementary cut uses anti-phase C18 preparative column to carry out gradient elution, the time that goes out peak according to fignal center is collected elutriant, underpressure distillation, to medicinal extract, obtains secondary cut, and secondary cut is dissolved in the volume that DMSO makes it to reach before secondary cut distillation and is marine microorganism fermentation broth sample;
2) preparation of His-DXR: take the full genome of Vibrio vulnificus as template, SEQ ID NO:1 and SEQ ID NO:2 are that primer carries out after pcr amplification, are cloned into pET-22b carrier and obtain recombinant plasmid pET22b-DXR; By inducing with IPTG after recombinant plasmid pET22b-DXR conversion intestinal bacteria, resuspended after centrifugal collection somatic cells, carry out ultrasonic degradation thalline, centrifugal collection supernatant, by supernatant process Ni 2+-NTA affinity chromatographic column wash-out, collects target protein Solution H is-DXR and is dissolved in dialyzate, with Amicon Ultra-4 Centrifugal Filter Devices centrifugal ultrafiltration desalination repeatedly, obtains His-DXR;
3) Screening of Antibacterial Activities: 175 μ L bacteria suspensions, 20 μ L resazurin solution and 5 μ L step 1) gained marine microorganism fermentation broth samples are joined in 96 microwell plates simultaneously; Set feminine gender and positive control simultaneously, in constant incubator, cultivate after 24 hours, the excitation wavelength in microplate reader is 530nm, and emission wavelength is that 590nm place measures its fluorescent value, and count out inhibiting rate by formula, filter out the cut of inhibiting rate more than 80% and stay and do the screening of living of next step enzyme;
Described bacteria suspension is wheel animalcule vibrios or bank formula vibrios or Vibrio vulnificus suspension; Negative control is: 5 μ L DMSO+175 μ L bacteria suspension+20 μ L resazurins; Positive control is: 5 μ L DMSO+175 μ L LB+20 μ L resazurins;
4) enzyme inhibition test screening alive: the cut that step 3) is filtered out is dissolved in DMSO and configures cut to be screened, is containing Tris-HCl damping fluid, MgCl 2dTT, NADPH and DXP, in the reaction solution of certain pH, add respectively cut to be screened, add step 2) gained His-DXR start reaction, under 340nm, measure light absorption value, arrange contrast and blank group, control group replaces cut to be screened with DMSO, blank group does not add His-DXR, in triplicate, calculates inhibiting rate; The cut of>=50% enzyme inhibiting rate alive is the 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor screening.
In described step 1), marine microorganism fermentation broth sample be prepared as seawater PDA liquid nutrient medium inoculated fungi, after cultivation as seed liquor; Rice solid fermentation substratum ferments, and after fungi fermentation maturation, adopts ethyl acetate soaking fermentation product, extracts meta-bolites, repeatedly extracts 3 times, soaks 24h at every turn; And adopt high-pressure emulsification cutting machine take ethyl acetate as solvent treatment tunning, and extracting intracellular organic matter, the liquid of extraction adopts Rotary Evaporators to be evaporated to medicinal extract, obtains elementary medicinal extract;
Elementary medicinal extract utilizes normal phase silicagel column, and normal hexane is set, and ethyl acetate ratio is carried out isocratic elution, by UV-detector detection signal, under 220nm, receives signal, and the time that goes out peak according to fignal center is collected elutriant, and elutriant concentrating under reduced pressure, obtains elementary cut;
Elementary cut uses anti-phase C18 preparative column to carry out the preparation of secondary cut, acetonitrile is set: water condition of gradient elution, by UV-detector detection signal, under 220nm, receive signal, the time that goes out peak according to fignal center is collected elutriant, underpressure distillation, to medicinal extract, obtains secondary cut, and secondary cut is dissolved in the volume that DMSO makes it to reach before secondary cut distillation and is marine microorganism fermentation broth sample.
Described step 2) in, take the full genome of Vibrio vulnificus E1758 as template, carry out PCR, reaction conditions with high-fidelity DNA polymerase Primerstar: 94 ℃ of sex change 30s; 53 ℃ of annealing 30s; 72 ℃ are extended 1.5min totally 30 circulations; PCR product reclaims through agarose gel electrophoresis, is cloned on pET-22b carrier, transforms bacillus coli DH 5 alpha competent cell, on the LB flat board that adds penbritin, cultivates 12h for 37 ℃; Picking list colony inoculation is in containing the LB substratum of penbritin, and 37 ℃ of joltings are spent the night; Build plasmid called after pET22b-DXR; By positive recombinant plasmid transformed coli strain BL21 (DE3); Picking list bacterium colony, is placed in 5ml LB (Amp+) substratum, and 37 ℃ of joltings are cultured to D600 and are about at 0.6 o'clock, and adding final concentration is 28 ℃ of inductions of IPTG of 0.5mmol/L, continues jolting 6h; The bacterium colony that has His-DXR expressing fusion protein is carried out; Bacterium liquid amplification induction, somatic cells carries out centrifugal collection 7800 rpm/min, after 4 ℃ of centrifugal 5min, is resuspended in 10 ml binding buffer liquid NPI-10, and add 1.5%Trioton × 100, and the DTT of 1mmol/L, the PMSF of 1mmol/L, at ultrasonic degradation thalline on ice, super 5s, stops 5s, 50% power; 4 ℃ of centrifugal collection supernatant 7800 rpm × 20min, proceed to supernatant in the Ni2+-NTA affinity chromatographic column of crossing through binding buffer liquid balance, at 4 ℃ light and slow rotation in conjunction with 60min after, abandoned stream fluid.With the lavation buffer solution NPI-20 of 10 times of Ni2+-NTA affinity chromatography column volumes; Washing Ni2+-NTA affinity chromatographic column, washes away non-specific binding albumen, and abandoned stream fluid, repeats once.Finally use the NPI-250 of 5 times of Ni2+-NTA affinity chromatography column volumes; Wash-out is also collected target protein Solution H is-DXR, by the new wash-out of albumen add-back duplicate removal again 1-3 time of collecting; The protein solution His-DXR collecting is dissolved in dialyzate, with Amicon Ultra-4 Centrifugal Filter Devices centrifugal ultrafiltration desalination repeatedly, obtains His-DXR;
Described binding buffer liquid NPI-10 is 50mmol/L NaH 2pO 4, 300mmol/L NaCl, 10mmol/L imidazoles, pH 8.0; Lavation buffer solution NPI-20 is 50mmol/L NaH 2pO 4, 300mmol/L NaCl, 20mmol/L imidazoles, pH 8.0; NPI-250 is 50mmol/L NaH 2pO 4, 300mmol/L NaCl, 250mmol/L imidazoles, pH 8.0; Dialyzate is 50mmol/LTris-HCl damping fluid, 2mmol/L DTT, and pH is 7.5.
Bacteria suspension described in described step 3) is 10 4the wheel animalcule vibrios or 10 of CFU/mL 4cFU/mL bank formula vibrios or 10 5cFU/mL Vibrio vulnificus; In the time being wheel animalcule vibrios and bank formula vibrios, resazurin solution is 500 μ g/mL, and in the time being Vibrio vulnificus, resazurin solution is 200 μ g/mL; Negative control is: 5 μ L DMSO+175 μ L bacteria suspension+20 μ L resazurins; Positive control is: 5 μ L DMSO+175 μ L LB+20 μ L resazurins.
Described step 4) is being for to contain 50mmol/LTris-HCl damping fluid, the MgCl of 3mmol/L 2, 2mmol/L DTT, the NADPH of 0.15mmol/L and the DXP of 0.25mmol/L, pH is in 7.5 100 μ l reaction solutions, under 37 degree, add respectively the cut to be screened of 5 μ l, adding step 2) gained His-DXR starts reaction, after 5min, under 340nm, measure light absorption value, arrange and contrast and blank group, control group replaces cut to be screened with DMSO, and blank group does not add His-DXR, in triplicate, calculate inhibiting rate; The cut of>=50% enzyme inhibiting rate alive is the screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor screening.
In described step 4), described temperature of reaction is 37 degree.
The present invention's 55 to 60 degree are optimal activity temperature of enzyme, and in the time that 37 spend, enzyme also has activity, and active substance is the not change of Yin Wendu and unknown change occurs also.
The method of key enzyme 1-deoxy-D-xylulose sugar-5 phosphoric acid racemases (Dxr) inhibitor in MEP approach (being isoprenoid and derivative route of synthesis thereof).MEP approach (being isoprenoid and derivative route of synthesis thereof) is most important in biological metabolism and Cell wall synthesis, it exists in some bacterium, protozoon and plant plastid, and not do not find in Mammals, be one of medicine target of antibacterial, antimalarial extremely likely and weedicide.Wherein, in this approach, one of the most key enzyme is 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase (1-deoxy-d-xylulose 5-phosphate reductoisomeraseDxr), generates MEP by oxidation NADPH catalysis 1-deoxy-D-xylulose sugar-5 phosphoric acid (1-deoxy-d-xylulose-5-phosphate DXP).We infer the compound that suppresses Dxr enzymic activity, will inevitably suppress to a certain extent the growth of bacterium.By Cloning and Expression Vibrio vulnificus Dxr albumen in intestinal bacteria and optimize its enzyme activity determination method, set up on this basis vibrios Dxr inhibitor high flux screening model, for finding that novel antibacterial medicine or lead compound take Dxr as target spot lay the foundation.
The embodiment of the present invention provides concrete screening step and method.Can find out, 21 activated cuts that the present invention obtains, finishing screen is chosen 6 cuts the inhibiting rate of His-DXR is greater than to 50%, illustrates that 6 cut samples of this marine microorganism fermented liquid have inhibition to His-DXR, and the cut screening is the inhibitor that the present invention needs.
The enzyme inhibition experiment alive of standard substance fosmidomycin (Fosmidomycin) is a positive control test.
Accompanying drawing explanation
Fig. 1 is the restriction enzyme digestion and electrophoresis figure of recombinant plasmid pET22b-DXR.
Fig. 2 is that SDS-PAGE analyzes His-DXR Expression and purification result figure.
Fig. 3 is different concns NADPH and OD(340nm) graph of a relation of absorption value.
Fig. 4 is the graph of a relation of NADP growing amount and time.
Fig. 5 A is that differing temps, 5B are that different pH values, 5C are different concns Mg 2+, the 5D figure that affect that are different concentration of substrate DXP on restructuring His-DXR catalyzed reaction.
Fig. 6 is the inhibiting rate figure of phosphamidon mycin different concns.
Embodiment
Describe embodiments of the invention below in detail, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has the element of identical or similar functions from start to finish.Be exemplary below by the embodiment being described with reference to the drawings, be intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
embodiment 1:from marine microorganism fermented liquid, screen 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor
Bacterial classification and plasmid
PET-22b carrier, bacillus coli DH 5 alpha, BL21(DE3) preserve by this laboratory, Vibrio vulnificus (MCCC E1758) wheel animalcule vibrios (MCCC E385), Vibrio campbellii (MCCC E333) is stored in Chinese Sea microbial strains preservation administrative center.
Main agents
Primer is synthetic by An Teao bio tech ltd, Xiamen, and DNA sequence dna is measured by Invitrogen company.
Restriction enzyme (Xho I, EcoR I), EcoR III ligase enzyme, high-fidelity DNA polymerase Primerstar, DL 2 kb DNA Marker are all purchased from TaKaRa company; Unstained Protein Molecular Weight Marker and BCA Protein Assay Kit are purchased from Thermo company of the U.S.; Sec.-propyl-β-D-sulfo-semi-lactosi (isopropyl-β-D-thiogalactoside, IPTG), dithiothreitol (DTT) (DL-Dithiothreitol, DTT), penbritin, sodium-chlor (NaCl) are purchased from the raw work in Shanghai; Ni 2+-NTA is affine resin is purchased from MACHEREY NAGEL company; 1-Deoxy-D-xylulose-5-phosphate sodium salt(DXP), reduced form β-Reduced nicotinamide-adenine dinucleotide 2 '-tetrasodium phosphate salt hydrate (NADPH), dimethyl sulfoxide (DMSO) (DMSO), two water disodium ethylene diamine tetraacetate (EDTA), Triton-X 100, benzene first sulphur fluorine (phenylmethylsulfonyl fluoride, PMSF), sodium laurylsulfonate (SDS) be purchased from Sigma company of the U.S.; Tryptone, Yeast Extract, Agar are purchased from OXOID company of Britain; Amicon Ultra-4 Centrifugal Filter Devices is purchased from Millipore company of the U.S., resazurin: traditional Chinese medicines chemical reagent company limited, article No.: 71035931)
Substratum
Testing bacterial classification activation medium used is LB substratum, and protein expression substratum is 2 × YT substratum, and adds as required penbritin (50 μ g/mL).
1, the preparation of marine microorganism fermentation broth sample:
Take thalassiomycetes as fermented bacterium, successively adopt solid fermentation, solvent extraction and silica gel chromatographic column obtain secondary cut;
1) adopt seawater PDA liquid nutrient medium; After high-temperature sterilization, strict aseptic technique, super clean bench inoculated fungi, 28 ℃ of shaking culture 2-3 days, using this culture as seed liquor;
2) adopt rice solid fermentation substratum to ferment, the bottled rice 105g of pyrometric cone of every 1L, millet 45g, adds 150 mLPDA nutrient solutions, and 121 ℃, 20min high-temperature sterilization, 15% inoculum size inoculation, room temperature is placed 28 days;
3) after fungi fermentation maturation, adopt ethyl acetate soaking fermentation product, extract meta-bolites, repeatedly extract 3 times, soak 24h at every turn.And adopt high-pressure emulsification cutting machine take ethyl acetate as solvent treatment tunning, and extracting intracellular organic matter, the liquid of extraction adopts Rotary Evaporators to be evaporated to medicinal extract, obtains elementary medicinal extract;
4) elementary medicinal extract utilizes normal phase silicagel column, and normal hexane is set, and ethyl acetate ratio is carried out isocratic elution, by UV-detector detection signal, under 220nm, receive signal, the time that goes out peak according to fignal center is collected elutriant, elutriant concentrating under reduced pressure, obtains elementary cut;
5) elementary cut uses anti-phase C18 preparative column to carry out the preparation of secondary cut, and acetonitrile is set: water condition of gradient elution, by UV-detector detection signal, under 220nm, receive signal, the time that goes out peak according to fignal center is collected elutriant, and underpressure distillation, to medicinal extract, obtains secondary cut;
6) cut equal-volume (equating with distillation front volume) is dissolved in to DMSO stand-by;
2, the preparation of His-DXR
1, the total genomic extraction of Vibrio vulnificus E1758 (with reference to Ai Delai bio tech ltd, Beijing---bacterial genomes DNA extraction test kit article No.: DN12)
1-deoxy-D-xylulose sugar-5 phosphoric acid racemases (1-Deoxy-D-xylulose-5-phosphate reductoisomerase) gene clone and order-checking
According to 16S comparison result on NCBI, Vibrio vulnificus (MCCC E1758) with vibrio vulnificuscMCP6 similarity reaches 99%, therefore according to vibrio vulnificuscMCP6 gene 1-deoxy-D-xylulose sugar-5 phosphoric acid racemases (hereinafter to be referred as DXR) sequences [NP_760741.1] design primers (D1 and D2), and add EcoR I, Xho I restriction enzyme site at upstream and downstream primer 5 ' end, primer sequence is as follows:
D1:5 '-TTCGGATCC gAATTCgATGCAAAAGCTAACGATTCT-3 ', underscore is EcoR I restriction enzyme site, sequence is shown in SEQ ID NO:1
D2:5 '-GTGGTGGTG cTCGAGtTTTGCAAGATAGTGGCG-3 ',, containing terminator codon, underscore is not Xho I restriction enzyme site.Sequence is shown in SEQ ID NO:2
Adopt conventional molecule clone technology, take the full genome of E1758 as template, carry out PCR, reaction conditions with high-fidelity DNA polymerase Primerstar: 94 ℃ of sex change 30s; 53 ℃ of annealing 30s; 72 ℃ are extended 1.5min totally 30 circulations.PCR product reclaims through agarose gel electrophoresis, is cloned on pET-22b carrier, transforms bacillus coli DH 5 alpha competent cell, on the LB flat board that adds penbritin, cultivates 12h for 37 ℃.Picking list colony inoculation is in containing the LB substratum of penbritin, and 37 ℃ of joltings are spent the night.In a small amount extract plasmid, carry out respectively PCR, the enzyme evaluation of cutting and check order.Build plasmid called after pET22b-DXR.Extract recombinant plasmid pET22b-DXR from transformant, take this plasmid of EcoR I-Xho I double digestion, obtain the DNA fragmentation of 1209bp, illustrate that DXR successfully inserts in pET-22b carrier (to the results are shown in Figure 1).Show the DXR and the known array (NP_760741.1) in full accord that insert by nucleotide sequencing result.
The prokaryotic expression of His-DXR fusion rotein and purifying
By positive recombinant plasmid transformed coli strain BL21 (DE3).Picking list bacterium colony, is placed in 5ml LB (Amp +) in substratum, 37 ℃ of joltings are cultured to D 600be about at 0.6 o'clock, adding final concentration is 28 ℃ of inductions of IPTG of 0.5mmol/L, continues jolting 6h.Utilize 12%SDS-PAGE to identify the bacterium colony of expressed fusion protein.The correct plasmid of restructuring is converted into e. coli bl21 (DE3), after IPTG induction, bacterial lysate obtains a protein band (the results are shown in Figure 2) to there being the bacterium colony of His-DXR expressing fusion protein to carry out (100ml) bacterium liquid amplification induction at about 44kD place, somatic cells carries out centrifugal collection 7800 rpm/min, after 4 ℃ of centrifugal 5min, be resuspended in 10 ml binding buffer liquid NPI-10 (50mmol/L NaH 2pO 4300mmol/L NaCl, 10mmol/L imidazoles, pH 8.0), and add 1.5%Trioton × 100, the DTT of 1mmol/L, the PMSF of 1mmol/L, at ultrasonic degradation thalline (super 5s on ice, stop 5s, 50% power), 4 ℃ of centrifugal collection supernatant 7800 rpm × 20min, proceed to supernatant the Ni crossing through binding buffer liquid balance 2+in-NTA affinity chromatographic column, at 4 ℃ light and slow rotation in conjunction with 60min after, abandoned stream fluid.With 10 times of Ni 2+the lavation buffer solution NPI-20(50mmol/L NaH of-NTA affinity chromatography column volume 2pO 4, 300mmol/L NaCl, 20mmol/L imidazoles, pH 8.0) and washing Ni 2+-NTA affinity chromatographic column, washes away non-specific binding albumen, and abandoned stream fluid, repeats once.Finally use 5 times of Ni 2+the NPI-250(50mmol/L NaH of-NTA affinity chromatography column volume 2pO 4, 300mmol/L NaCl, 250mmol/L imidazoles, pH 8.0) and wash-out collect target protein Solution H is-DXR, by the new wash-out of albumen add-back duplicate removal again of collecting 1-3 time.The protein solution His-DXR collecting is dissolved in dialyzate (50mmol/LTris-HCl damping fluid, 2mmol/L DTT, pH is 7.5) in, with Amicon Ultra-4 Centrifugal Filter Devices centrifugal ultrafiltration desalination repeatedly, with 12% SDS-PAGE qualification result.
The determination of activity of 1-deoxy-D-xylulose sugar-5 phosphoric acid racemases (His-DXR)
In view of NADPH and NADP+ 340 and 260nm place have maximum absorption band separately, therefore take NADPH as coenzyme by measuring the variation of 340nm light absorption value, the activity of quantitative assay enzyme.With 340nm light absorption value mapping (Fig. 3) corresponding to the NADPH of different concns, the OD measuring in corresponding experiment 340value can calculate according to mapped linear relationship the growing amount of NADP.His-DXR is at metal ions M g 2+effect under catalysis DXP generate 2-C-methyl-D-erythritol 4-phosphate (MEP) and make NADPH be oxidized to NADP, the reduction of measuring 340nm place light absorption value can reflect the activity of His-DXR.In experimental group and blank group, reaction system is 100 μ l, wherein all contains 50mmol/LTris-HCl damping fluid, the MgCl of 3mmol/L 2, 2mmol/L DTT, the NADPH of 0.15mmol/L and the DXP of 0.5mM, pH is 7.5.Be (i.e. 2.9 μ g) enzyme (His-DXR) of 0.58ug/ul to adding 5ul concentration in experimental group, reaction at 37 ℃, measures the variation of 340nm place light absorption value, and every 1min reads once, with NADP growing amount, the reaction times is mapped, the maximum value of getting △ NADP/min represents the size of enzyme activity; In blank group, do not add His-DXR enzyme, every 1min reads the OD of a NADPH 340, the variation mapping (Fig. 4) according to the passing of time to its absorption value;
Determination of activity result is as follows:
The 340nm light absorption value corresponding to NADPH of different concns made graph discovery linear (y=1.660x+0.0537, R 2=0.9915), see Fig. 3.According to the OD measuring in reaction 340value can calculate the variation of concentration before and after NADPH reaction, thereby can calculate the growing amount of NADP, thereby determine the vigor of His-DXR;
To restructuring, His-DXR carries out active detection assay, result shows that substrate DXP is under the condition of NADPH existence, can make NADPH be oxidized to NADP, every 1min measures the light absorption value of NADPH at 340nm place, with NADP growing amount to reaction times mapping (the results are shown in Figure 4), can find the growing amount of the NADP in 5min and reaction times in good linear relation (y=0.0228x,, R 2=0.999), its enzyme activity is 0.0228 mmol/Lmin.So the reaction times should be chosen in 5min, the enzyme of mensuration speed of reaction alive is the maximum speed of reaction in the sufficient situation of substrate.Blank group NADPH is not enzyme-added in the situation that, and along with its OD340 light absorption value of time lapse does not change substantially, NADPH its amount in 5min can not change, and can not affect the mensuration that enzyme is lived;
To restructuring, His-DXR carries out active detection assay, result shows that substrate DXP is under the condition of NADPH existence, can make NADPH be oxidized to NADP, every 2min measures the light absorption value of NADPH at 340nm place, with NADP growing amount, the reaction times is mapped, can find that the growing amount of the NADP in 8min and reaction times are in good linear relation, reaction times exceedes after 18min, the growing amount curve of NADP is obviously tending towards straight and no longer rises, illustrate that reaction has reached whole end, data after this should not be re-used as the foundation that statistics NADP growing amount changes.So the reaction times should be chosen in 5min, the enzyme of mensuration speed of reaction alive is the maximum speed of reaction in the sufficient situation of substrate;
For the condition determination of optimum combination His-DXR enzymic activity, respectively to differing temps, different pH value and different concns substrate DXP, Mg 2+on enzymic activity, impact is studied, and result shows that the optimal reactive temperature of His-DXR is 55-60 ℃ (Fig. 5 A), optimal reaction pH value 7.5-8(Fig. 5 B), Mg 2+optimum concn be 3mmol/L(Fig. 5 C), the optimal concentration of DXP is 0.25mmol/L(Fig. 5 D);
The mensuration of enzymatic property
1) optimal reactive temperature: within the scope of 32 ~ 68 ℃, containing 50mmol/LTris-HCl damping fluid, the MgCl of 3mmol/L 2, 2mmol/L DTT, the NADPH of 0.15mmol/L and the DXP of 0.5mmol/L, measure respectively enzyme and live in the reaction solution that pH is 7.5.With substrate NADP growing amount, temperature is mapped.See Fig. 5 A;
2) optimal reaction pH value: at 37 ℃, measure respectively enzyme and live under different pH conditions, with NADP growing amount, pH value in reaction is mapped, contain the Tris of 50mmol/L in reaction solution used, the MgCl of 3mmol/L 2, 2mmol/L DTT, the NADPH of 0.15mmol/L and the DXP of 0.5mmol/L, regulating pH value with HCl is 3 ~ 10.See Fig. 5 B;
3) the suitableeest concentration of metal ions: at different Mg 2+under concentration, measure respectively enzyme live, with NADP growing amount to Mg 2+concentration is mapped, and contains the Tris of 50mmol/L, 2mmol/L DTT, the Mg of the NADPH of 0.15mmol/L and the DXP of 0.5mmol/L and 0 ~ 8mmol/L different concns in reaction solution used 2+, pH is 7.5.See Fig. 5 C;
4) mensuration of concentration of substrate: measure the enzyme of substrate DXP in the time of different concns at 37 ℃ and live, with the growing amount of NADP, the concentration of substrate DXP is mapped.In reaction solution used, contain 50mmol/LTris-HCl damping fluid, the MgCl of 3mmol/L 2, 2mmol/L DTT, the NADPH of 0.15mmol/L and the DXP of 0 ~ 0.5mmol/L, pH is 7.5.See Fig. 5 D;
3, Screening of Antibacterial Activities:
Adopt the method for resazurin development process as detection of active, resazurin colour developing ratio juris is: the serum lactic dehydrogenase in viable cell can be converted into resazurin (blueness) fluorescent substance resorufin (pink redness), producing fluorescent signal, is that 530nm, emission wavelength are fluorescent value can be detected under 590nm in excitation wavelength.Resorufin can continue to be reduced to non-blooming material dihydro resorufin (white) by cell, fluorescent signal is declined, non-activity or dead cell has been lost metabolic capacity and can not have been reduced resazurin, also just can not produce fluorescent signal, so this method energy specific detection active cells;
Experiment adopts 96 orifice plates operations, and the inhibiting rate calculating according to the color of 96 orifice bores and fluorescent value judges whether material has bacteriostatic activity.In hole, color is blue, and representative sample has and suppresses active aimed strain; Color pinkiness or redness in 96 holes, representative sample does not suppress active to aimed strain.The calculation formula of inhibiting rate formula is
Inhibiting rate %=100%-(experimental port fluorescent value-positive control fluorescent value)/(negative control fluorescent value-positive control fluorescent value) × 100%.Inhibiting rate is more than 80%, and representative sample has stronger restraining effect to aimed strain;
Experimental strain wheel animalcule vibrios (MCCC E385), Vibrio campbellii (MCCC E333), Vibrio vulnificus (MCCC E1758) is located away from Zhanjiang and infects prawn, is stored in 30% glycerine, and-80 ℃ are frozen.Three strain bacterium are inoculated in respectively in 5mL LB nutrient solution, after 3 hours, collect thalline in 37 ℃ of shaking culture, use 0.9% normal saline dilution, with blood counting chamber counting, adjusting bacteria suspension concentration with the dilution of LB nutrient solution is 10 4cFU/mL(wheel animalcule vibrios and Kan Shi vibrios) and 10 5cFU/mL(Vibrio vulnificus);
With sterilized water configuration resazurin concentration be the mother liquor of 5000 μ g/mL, and with the membrane filtration packing of 0.22 μ m, frozen at-20 ℃.Getting that a pipe dilutes it is respectively that 500 μ g/mL(are applicable to wheel animalcule vibrios and bank formula vibrios) and 200 μ g/mL(be applicable to Vibrio vulnificus);
The cut of 175 μ L bacteria suspensions, 20 μ L resazurin solution and 5 μ L steps 1 is joined in 96 microwell plates simultaneously; In 96 orifice plates, set negative control and positive control.(negative control is: 5 μ L DMSO+175 μ L bacteria suspension+20 μ L resazurins; Positive control is: 5 μ L DMSO+175 μ L LB+20 μ L resazurins), 96 orifice plates are put in 37 ℃ of constant incubators and cultivated 24h, after 24h, observe the color in each hole in 96 microwell plates, and by 96 orifice plates put into microplate reader excitation wavelength be 530nm emission wavelength be 590nm place measure fluorescent value and count out inhibiting rate by formula.The cut of inhibiting rate more than 80% stays and does the screening alive of next step enzyme;
4, enzyme inhibition test screening alive:
(1) enzyme of standard substance fosmidomycin (Fosmidomycin) is lived and is suppressed experiment
Fosmidomycin is dissolved in DMSO, being made into concentration is 50 μ mol/L mother liquors, become by twice gradient dilution: 25 μ mol/L, 12.5 μ mol/L, 6.25 μ mol/L, 3.125 μ mol/L, contain 50mmol/LTris-HCl damping fluid in 5 holes, the MgCl2 of 3mmol/L, 2mmol/L DTT, the NADPH of 0.15mmol/L and the DXP of 0.25mmol/L, pH is in 7.5 100 μ l reaction solutions, add respectively the fosmidomycin 5 μ l of each concentration, in 100 μ l systems, the concentration of fosmidomycin is 2500nmol/L, 1250nmol/L, 625nmol/L, 312.5nmol/L, 156.25nmol/L.Then adding respectively step 2 gained concentration is the i.e. 2.9 μ g of 0.58ug/ul() His-DXR 5ul starts reaction.After 5min, under 340nm, measure light absorption value.Arrange and contrast and blank group, control group replaces fosmidomycin with DMSO, and blank group not enzyme-added (not adding His-DXR) in triplicate, is calculated inhibiting rate.Inhibiting rate (%)=(sample light absorption value-contrast light absorption value)/(blank absorbency-contrast light absorption value) × 100%
Fosmidomycin, because of its structure and enzyme reaction substrate DXP alive isomerization product structural similitude, suppresses therefore be considered to specificity
The microbiotic of 1-deoxy-D-xylulose sugar-5 phosphoric acid racemases (DXR).Map and calculate phosphine amine with GraphPad prism software
Mycin is for the IC of Vibrio vulnificus 1-deoxy-D-xylulose sugar-5 phosphoric acid racemases (DXR) 50(503nhibiting concentration) is 390nmol/L,
See Fig. 6;
(2) marine microorganism fermentation broth sample enzyme is lived and is suppressed experiment
The cut of bacteriostatic activity >=80% is pressed to standard substance fosmidomycin enzyme experimental technique and the inhibiting rate method of calculation of suppressing alive, detect the inhibition activity of testing sample, >=50% enzyme inhibiting rate alive is considered as activated cut.21 cuts have been screened in this experiment altogether, and inhibiting rate is in table 1 and 2, and wherein 6 cuts are effective.
The His-DXR inhibitor screening model that utilizes aforesaid method to build, screens activated 21 microbial fermenters that filter out by resazurin method, is greater than 50% determines positive findings according to inhibiting rate.
The inhibiting rate of 1:6 cut of table
The inhibiting rate of 2:15 cut of table
Cut label Experimental group Control group Blank group Inhibiting rate
236-12 0.22 0225. 0.311 0.00%
236-13 0.235 0225. 0.311 11.40%
236-14 0.232 0.225 0.311 7.89%
236-15 0.238 0.225 0.311 14.91%
236-18 0.225 0.225 0.311 0.00%
235-27 0.263 0.225 0.311 44.30%
236-46-1 0.258 0.225 0.311 37.97%
236-46-2 0.254 0.225 0.311 34.18%
236-46-3 0.263 0.225 0.311 44.03%
236-46-6 0.232 0.225 0.311 8.05%
236-60 0.237 0.225 0.311 13.79%
236-83 0.248 0.225 0.311 26.44%
251-18 0.222 0.212 0.324 9.02%
251-31-1 0.231 0.212 0.324 17.21%
251-31-2-1 0.215 0.212 0.324 2.46%
In table 1 and table 2,251 represent sharp knife Fusariumsp fermentation gained cut; 236 represent the mould fermentation gained of white side tooth cut.
Experimental group is: NADPH+DXP+ cut+reaction solution+enzyme DXR;
Control group is: NADPH+DXP+DMSO+ reaction solution+enzyme DXR;
Blank group is: NADPH+DXP+DMSO+ reaction solution;
As can be seen from Table 1,6 cuts in table are greater than 50% to the inhibiting rate of His-DXR, illustrate that 6 cut samples of this marine microorganism fermented liquid have inhibition to His-DXR, and the cut screening is the inhibitor that the present invention needs.
As can be seen from Table 2,15 cuts in table are less than 50% to the inhibiting rate of His-DXR.15 cut samples that this marine microorganism fermented liquid is described are not obvious to the inhibition of His-DXR, and the cut screening is not the inhibitor that the present invention needs.
Although illustrated and described embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention in the situation that not departing from principle of the present invention and aim, modification, replacement and modification.
SEQUENCE LISTING
<110> State Oceanic Administration Bureau The Third Oceanography Institute
<120> method of screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor from marine microorganism fermented liquid
<130> P9738
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 36
<212> DNA
<213> synthetic
<400> 1
ttcggatccg aattcgatgc aaaagctaac gattct 36
<210> 2
<211> 33
<212> DNA
<213> synthetic
<400> 2
gtggtggtgc tcgagttttg caagatagtg gcg 33

Claims (5)

1. a method of screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor from marine microorganism fermented liquid, is characterized in that, comprises the steps,
1) preparation of marine microorganism fermentation broth sample: seawater PDA liquid nutrient medium inoculated fungi, after cultivation, continue fermentation as seed liquor, after fungi fermentation maturation, extract meta-bolites; And extract intracellular organic matter, the liquid concentration of extraction, to medicinal extract, obtains elementary medicinal extract;
Elementary medicinal extract utilizes normal phase silicagel column to carry out isocratic elution, and the time that goes out peak according to fignal center is collected elutriant, and concentrating under reduced pressure, obtains elementary cut;
Elementary cut uses anti-phase C18 preparative column to carry out gradient elution, the time that goes out peak according to fignal center is collected elutriant, underpressure distillation, to medicinal extract, obtains secondary cut, and secondary cut is dissolved in the volume that DMSO makes it to reach before secondary cut distillation and is marine microorganism fermentation broth sample;
2) preparation of His-DXR: take the full genome of Vibrio vulnificus as template, SEQ ID NO:1 and SEQ ID NO:2 are that primer carries out after pcr amplification, are cloned into pET-22b carrier and obtain recombinant plasmid pET22b-DXR; By inducing with IPTG after recombinant plasmid pET22b-DXR conversion intestinal bacteria, resuspended after centrifugal collection somatic cells, carry out ultrasonic degradation thalline, centrifugal collection supernatant, by supernatant process Ni 2+-NTA affinity chromatographic column wash-out, collects target protein Solution H is-DXR and is dissolved in dialyzate, with Amicon Ultra-4 Centrifugal Filter Devices centrifugal ultrafiltration desalination repeatedly, obtains His-DXR;
3) Screening of Antibacterial Activities: 175 μ L bacteria suspensions, 20 μ L resazurin solution and 5 μ L step 1) gained marine microorganism fermentation broth samples are joined in 96 microwell plates simultaneously; Set feminine gender and positive control simultaneously, in constant incubator, cultivate after 24 hours, the excitation wavelength in microplate reader is 530nm, and emission wavelength is that 590nm place measures its fluorescent value, and count out inhibiting rate by formula, filter out the cut of inhibiting rate more than 80% and stay and do the screening of living of next step enzyme;
Described bacteria suspension is wheel animalcule vibrios or bank formula vibrios or Vibrio vulnificus suspension; Negative control is: 5 μ L DMSO+175 μ L bacteria suspension+20 μ L resazurins; Positive control is: 5 μ L DMSO+175 μ L LB+20 μ L resazurins;
4) enzyme inhibition test screening alive: the cut that step 3) is filtered out is dissolved in DMSO and configures cut to be screened, is containing Tris-HCl damping fluid, MgCl 2dTT, NADPH and DXP, in the reaction solution of certain pH, add respectively cut to be screened, add step 2) gained His-DXR start reaction, under 340nm, measure light absorption value, arrange contrast and blank group, control group replaces cut to be screened with DMSO, blank group does not add His-DXR, in triplicate, calculates inhibiting rate; The cut of>=50% enzyme inhibiting rate alive is the 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor screening.
2. described in claim 1, from marine microorganism fermented liquid, screen the method for 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor, it is characterized in that, in described step 1), marine microorganism fermentation broth sample be prepared as seawater PDA liquid nutrient medium inoculated fungi, after cultivation as seed liquor; Rice solid fermentation substratum ferments, and after fungi fermentation maturation, adopts ethyl acetate soaking fermentation product, extracts meta-bolites, repeatedly extracts 3 times, soaks 24h at every turn; And adopt high-pressure emulsification cutting machine take ethyl acetate as solvent treatment tunning, and extracting intracellular organic matter, the liquid of extraction adopts Rotary Evaporators to be evaporated to medicinal extract, obtains elementary medicinal extract;
Elementary medicinal extract utilizes normal phase silicagel column, and normal hexane is set, and ethyl acetate ratio is carried out isocratic elution, by UV-detector detection signal, under 220nm, receives signal, and the time that goes out peak according to fignal center is collected elutriant, and elutriant concentrating under reduced pressure, obtains elementary cut;
Elementary cut uses anti-phase C18 preparative column to carry out the preparation of secondary cut, acetonitrile is set: water condition of gradient elution, by UV-detector detection signal, under 220nm, receive signal, the time that goes out peak according to fignal center is collected elutriant, underpressure distillation, to medicinal extract, obtains secondary cut, and secondary cut is dissolved in the volume that DMSO makes it to reach before secondary cut distillation and is marine microorganism fermentation broth sample.
3. described in claim 1, from marine microorganism fermented liquid, screen the method for 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor, it is characterized in that, described step 2) in, take the full genome of Vibrio vulnificus E1758 as template, carry out PCR, reaction conditions with high-fidelity DNA polymerase Primerstar: 94 ℃ of sex change 30s; 53 ℃ of annealing 30s; 72 ℃ are extended 1.5min totally 30 circulations; PCR product reclaims through agarose gel electrophoresis, is cloned on pET-22b carrier, transforms bacillus coli DH 5 alpha competent cell, on the LB flat board that adds penbritin, cultivates 12h for 37 ℃; Picking list colony inoculation is in containing the LB substratum of penbritin, and 37 ℃ of joltings are spent the night; Build plasmid called after pET22b-DXR; By positive recombinant plasmid transformed coli strain BL21 (DE3); Picking list bacterium colony, is placed in 5ml LB (Amp+) substratum, and 37 ℃ of joltings are cultured to D600 and are about at 0.6 o'clock, and adding final concentration is 28 ℃ of inductions of IPTG of 0.5mmol/L, continues jolting 6h; The bacterium colony that has His-DXR expressing fusion protein is carried out; Bacterium liquid amplification induction, somatic cells carries out centrifugal collection 7800 rpm/min, after 4 ℃ of centrifugal 5min, is resuspended in 10 ml binding buffer liquid NPI-10, and add 1.5%Trioton × 100, and the DTT of 1mmol/L, the PMSF of 1mmol/L, at ultrasonic degradation thalline on ice, super 5s, stops 5s, 50% power; 4 ℃ of centrifugal collection supernatant 7800 rpm × 20min, proceed to supernatant in the Ni2+-NTA affinity chromatographic column of crossing through binding buffer liquid balance, at 4 ℃ light and slow rotation in conjunction with 60min after, abandoned stream fluid;
With the lavation buffer solution NPI-20 of 10 times of Ni2+-NTA affinity chromatography column volumes; Washing Ni2+-NTA affinity chromatographic column, washes away non-specific binding albumen, and abandoned stream fluid, repeats once;
Finally use the NPI-250 of 5 times of Ni2+-NTA affinity chromatography column volumes; Wash-out is also collected target protein Solution H is-DXR, by the new wash-out of albumen add-back duplicate removal again 1-3 time of collecting; The protein solution His-DXR collecting is dissolved in dialyzate, with Amicon Ultra-4 Centrifugal Filter Devices centrifugal ultrafiltration desalination repeatedly, obtains His-DXR;
Described binding buffer liquid NPI-10 is 50mmol/L NaH 2pO 4, 300mmol/L NaCl, 10mmol/L imidazoles, pH 8.0; Lavation buffer solution NPI-20 is 50mmol/L NaH 2pO 4, 300mmol/L NaCl, 20mmol/L imidazoles, pH 8.0; NPI-250 is 50mmol/L NaH 2pO 4, 300mmol/L NaCl, 250mmol/L imidazoles, pH 8.0; Dialyzate is 50mmol/LTris-HCl damping fluid, 2mmol/L DTT, and pH is 7.5.
4. the method for screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor described in claim 1 from marine microorganism fermented liquid, is characterized in that, bacteria suspension described in described step 3) is 10 4the wheel animalcule vibrios or 10 of CFU/mL 4cFU/mL bank formula vibrios or 10 5cFU/mL Vibrio vulnificus; In the time being wheel animalcule vibrios and bank formula vibrios, resazurin solution is 500 μ g/mL, and in the time being Vibrio vulnificus, resazurin solution is 200 μ g/mL; Negative control is: 5 μ L DMSO+175 μ L bacteria suspension+20 μ L resazurins; Positive control is: 5 μ L DMSO+175 μ L LB+20 μ L resazurins.
5. described in claim 1, from marine microorganism fermented liquid, screen the method for 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor, it is characterized in that, described step 4) is that the cut that step 3) is filtered out is dissolved in DMSO, is containing 50mmol/LTris-HCl damping fluid, the MgCl of 3mmol/L 2, 2mmol/L DTT, the NADPH of 0.15mmol/L and the DXP of 0.25mmol/L, pH is in 7.5 100 μ l reaction solutions, under 37 degree, add respectively 5 μ l cut to be screened, adding step 2) gained His-DXR starts reaction, after 5min, under 340nm, measure light absorption value, arrange and contrast and blank group, control group replaces cut to be screened with DMSO, and blank group does not add His-DXR, in triplicate, calculate inhibiting rate; The cut of>=50% enzyme inhibiting rate alive is the screening 1-deoxy-D-xylulose sugar-5 phosphoric acid racemase inhibitor screening.
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